RU2312891C1 - Bioemulgator-producing association microorganism strains for degradation of petroleum and petroleum products in soil, fresh and sea water - Google Patents

Abstract

FIELD: biotechnology, microbiology, ecology.

SUBSTANCE: invention proposes the association of strains of microorganisms Rhodococcus sp. BKM Ac-2532D, Rhodococcus sp. BKM Ac-2533D, Pseudomonas sp. BKM B-2376D and Pseudomonas putida BKM B-2380D producing bioemulgators used in biodegradation of petroleum and petroleum products in soils, fresh and sea water. Using the association of these strains provides carrying out degradation of petroleum and petroleum products at the increased salt being both at normal and low temperature. Invention can be used in treatment of soils, water and marine water landing area against pollution with petroleum and petroleum products.

EFFECT: valuable biological and ecological properties of association.

2 tbl, 1 dwg, 3 ex

Description

The invention relates to the field of biotechnology and can be used for the degradation of oil and oil products, in particular oil hydrocarbons, in the treatment of soils, water and marine waters from pollution by oil and oil products.

A known consortium of oil-oxidizing microorganisms Pseudomonas putida PI Ko-1, Pseudomonas ftuorescens PI-896, Micrococcus sp. PI Ku-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1, which is used as a biological product to combat soil pollution with oil and oil products (RU 2191643, class B09C 1/10, 10.27.2002).

The disadvantage of the association is the inability to degrade oil and oil products at temperatures below 12 ° C.

Known biological product for cleaning environmental objects from pollution by oil and oil products, including the association of aerobic bacterial cultures Acidovorax delafleldii HT-1, Burkholderia caryophylii HT-4, Pseudomonas fluorescens biovar II HT-6, Citrobacter amalonaticus Jan-3-2, Pseudomonas 8-2, Serratia marcescens Jan-10-2 with a titer of living cells of at least 10 ¹⁰ .

The disadvantage of the association is the inability to degrade oil and oil products in the presence of an increased salt concentration (3-5%).

An association is known consisting of R-dissociants of strains of Rhodococcus ruber VKM Ac-1513D and Rhodococcus erythropolis VKM Ac-1514D, which degrades oil hydrocarbons in a wide range of salinity (0.05-10%) (optimal salinity of the medium is 3-5%) and temperatures (8-35 ° С) (optimum temperature is 25-27 ° С). This association is the basis of the biological product "Roder" (RU 2174496, CL C02F 3/34, 10.10.2001).

The disadvantage of microorganisms is the inability to produce bioemulsifiers (surfactants of natural origin), which increase the efficiency of biodegradation of difficultly decomposed oil fractions.

A known association of microorganisms includes the following microorganisms: Ralstonia eutropha B2 (FERM P-16672), Aureobasidium pullulans B10 (FERM P-16674), Ralstonia eutropha T23 (FERM P-16671), Burkholderia pyrrocinia T26 (FERM P-16669) 3 (FERM P-16673), Burkholderia pyrrocinia × 4 (FERM P-16670), which can be used to clean contaminated areas from oil and oil products (JP 11266858, 10/05/1999).

The disadvantage of the association is the ability to degrade only monoaromatic hydrocarbons (toluene, xylene, benzene) that make up the oil and the inability to degrade polyaromatic hydrocarbons.

The objective of this invention is to obtain an association of bacterial strains producing bioemulsifiers and capable of degradation of oil and oil products in soil, fresh and sea water at high salt concentrations.

The technical effect of using the proposed association is that the degradation of oil and oil products is carried out at an increased salt concentration both at normal and low temperatures, in addition, the bacterial strains that are part of the association produce surfactants (bioemulsifiers).

An association of bacteria of the genera Rhodococcus and Pseudomonas is proposed, including the following strains of bacteria Rhodococcus sp. BKM Ac-2532D, Rhodococcus sp. BKM Ac-2533D, Pseudomonas sp. BKM B-2387D and Pseudomonas putida BKM B-2380D, producing bioemulsifiers, for biodegradation of oil and oil products in soils, fresh and sea water.

It is intended for biodegradation of oil and oil products in soils, fresh and sea water both at normal and at low temperatures. Bacterial strains are contained in the association in equal amounts.

The bacterial strains that make up the association are deposited in the All-Russian Collection of Microorganisms (BKM) under the following numbers:

Rhodococcus sp. X5 - BKM Ac-2532D,

Rhodococcus sp. S67 - BKM Ac-2533D,

Pseudomonas sp. BS3962 - BKM B-2387D,

Pseudomonas putida BS3961 - BKM B-2380D.

Identification of bacterial strains was carried out by determining the partial nucleotide sequence of 16S rRNA genes.

The microorganisms that make up the association are sensitive to the following antibiotics: ampicillin, tetracycline, nalidixic acid, erythromycin, novobiocin, streptomycin, kanamycin, claforan, rifampicin, chloramphenicol.

The bacterial strain Rhodococcus sp. BKM Ac-2532D is isolated from the wastewater of a rubber plant.

Cultural and morphological characters.

Gram-positive straight, curved rods 0.7-0.8 × 1.6-2.8 microns in size, often the culture breaks up into coccal forms. The culture is motionless. On standard nutrient media (MPA, LB), after 48 hours it forms small, rough cream-colored colonies.

Physiological and biochemical characteristics.

Aerobe. It grows in the temperature range from 4 ° C to 30 ° C, the optimum is 25 ° C. The optimum pH for growth is 6.0-8.0. Capable of growing in the presence of 3% NaCl. The oxidase test is negative. Catalase test is positive. It uses oil, petroleum products, and individual hydrocarbons as a source of growth. The microorganism does not need additional growth factors.

The bacterial strain Rhodococcus sp. BKM Ac-2533D is isolated from the soil from the territory of a gas station.

Cultural and morphological characters.

Gram-positive straight, curved rods 0.7-0.8 × 1.6-2.8 microns in size, often the culture breaks up into coccal forms. The culture is motionless. On standard nutrient media (MPA, LB), after 48 hours it forms small, rough cream-colored colonies.

Physiological and biochemical characteristics.

Aerobe. It grows in the temperature range from 4 ° C to 30 ° C, the optimum is 25 ° C. The optimum pH for growth is 6.0-8.0. Capable of growing in the presence of 3% NaCl. The oxidase test is negative. Catalase test is positive. It uses oil, petroleum products, and individual hydrocarbons as a source of growth. The microorganism does not need additional growth factors.

The bacterial strain Pseudomonas sp. BKM B-2387D is isolated from soil contaminated with creosote.

Cultural and morphological characters.

Moving straight or curved sticks 0.8-0.9 × 1.5-3 microns in size. On standard nutrient media (MPA, LB, King B) it forms round colonies 2-3 mm in diameter, flat, smooth, the edges are slightly uneven, white, opaque.

Gram stain and Gram reaction by the Gregersen method (1978) are gram-negative.

Physiological and biochemical characteristics.

Aerobe. Hemorganotroph. Does not need growth factors. The oxidase test is positive. The range test is positive. It forms a fluorescent pigment. It does not grow at + 41 ° C. It does not have the ability to denitrify to form nitrogen. Gelatin does not hydrolyze. Starch does not hydrolyze. Uses glucose, glutamate, succinate as the sole carbon source. It uses benzoate, salicylate, naphthalene, phenanthrene, oil and diesel fuel as a growth source. The optimum pH for growth is 6.0-8.0. Capable of growing in the presence of 3% NaCl.

Genetic signs.

The microorganism contains a conjugative plasmid for biodegradation of PAHs pNF142 of 90 kbp. The presence of a conjugative plasmid containing PAH biodegradation genes increases the degradation potential due to the distribution of genes among indigenous microorganisms.

The bacterial strain Pseudomonas putida BKM B-2380D was isolated from the treatment plants of a coke oven gas plant.

Cultural and morphological characters.

Moving straight or curved sticks 0.8-0.9 × 1.5-3 microns in size. On standard nutrient media (MPA, LB, King B) it forms round colonies 2-3 mm in diameter, flat, smooth, the edges are slightly uneven, white, opaque.

Gram stain and Gram reaction by the Gregersen method (1978) are gram-negative.

Physiological and biochemical characteristics.

Aerobe. Hemorganotroph. Does not need growth factors. The oxidase test is positive. Catalase test is positive. It forms a fluorescent pigment. It does not grow at + 41 ° C. It does not have the ability to denitrify to form nitrogen. Gelatin does not hydrolyze. Starch does not hydrolyze. Uses glucose, glutamate, succinate as the sole carbon source. It uses benzoate, salicylate, naphthalene, phenanthrene, oil and diesel fuel as a growth source. The optimum pH for growth is 6.0-8.0. Capable of growing in the presence of 3% NaCl.

Genetic signs.

The microorganism contains two plasmids in the size of 100 and 50 TPN. Plasmid pBS1141 with a size of 100 kb contains genes for biodegradation of naphthalene and phenanthrene, is conjugative and belongs to the P9 incompatibility group. The presence of a conjugative plasmid containing PAH biodegradation genes in the bacterial strain Pseudomonas putida BKM B-2380D contributes to an increase in degradation potential due to the distribution of genes among indigenous microorganisms.

Plasmid pBS1142 with a size of 50 TPN is cryptic and does not contain biodegradation genes.

The selected microorganisms that make up the association are capable of utilizing diesel fuel, oil, fuel oil or polycyclic aromatic hydrocarbons as the sole source of carbon and energy at temperatures from 4 ° C to 32 ° C.

A study of the ability of microorganisms to grow on oil and diesel fuel in the presence of 3% NaCl at 24 ° C and 4-6 ° C showed (Table 2) that in the presence of 3% NaCl microorganisms grow better on oil than on diesel fuel at 24 ° C and 4-6 ° C, i.e. capable of degrading not only light but also heavy oil fractions.

When grown on hexadecane, these microorganisms produce bioemulsifiers that increase the efficiency of bio-utilization of oil and oil products. The study of the degree of oil degradation in a liquid mineral medium showed that individual bacterial strains are able to degrade from 15% to 26% of oil at 24 ° C for 7-10 days and from 28% to 47% at 4-6 ° C for 10- 20 days (see drawing).

Example 1

Bacterial strains are grown in Erlenmeyer flasks with 100 ml of Evans minimum medium containing (g / l): K ₂ HPO ₄ - 8.71 g; 5 M solution of NH ₄ Cl - 1 ml; 0.1 M solution of Na ₂ SO ₄ - 1 ml; 62 mm solution of MgCl ₂ - 1 ml; 1 mm solution of CaCl ₂ - 1 ml; 0.005 mM solution of (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O - 1 ml; trace elements - 1 ml (trace element composition, g / l: ZnO - 0.41 g; FeCl ₃ · 6H ₂ O -5.4 g; MnCl ₂ · 4H ₂ O - 2 g; CuCl ₂ · 2H ₂ O - 0, 17 g; CoCl ₂ · O - 0.48 g; H ₃ BO ₃ - 0.06 g; pH 7) with the addition of oil to a final concentration of 1.65% by weight. Inoculation of flasks with oil is carried out with a suspension of microorganisms (10 ml per 100 ml of medium, inoculative dose of 1-5 × 10 ⁷ cells / ml). After seeding, the flasks are placed on a circular rocking chair (120 rpm) and grown for 7-10 days at a temperature of 24 ° C and for 10-20 days at a temperature of 4-6 ° C.

An uninoculated flask with medium and oil is used as a control. The degree of oil degradation is determined using the gravimetric method. In 4 selected microorganisms, the degree of oil degradation at 4-6 ° C is higher (28-46%) than at 24 ° C (15-26%).

Example 2

The cultivation of the association of microorganisms and inoculation is performed as in Example 1. The biomass of the bacterial strains that make up the association is taken in equal amounts, based on the assumption that the seed dose to the flask is 1-5 × 10 ⁷ cells / ml. The cultivation of the association is carried out at 4-6 ° C for 10, 20 and 30 days. The degree of oil degradation, determined using the gravimetric method, ranges from 20% to 45%. The degree of oil degradation increases with increasing experiment duration up to 30 days. The data obtained confirm the assumption that for more effective degradation of oil in a liquid mineral medium, it is necessary to increase the experiment time to 30 days.

Example 3

The emulsifying activity of bacterial strains is determined visually according to the Broderic and Cooney method on a four-point scale and by changing the optical density of the supernatant with hexadecane according to the Cirigliano and Carman method in our modification. 1 ml of culture grown on Evans medium with hexadecane was centrifuged for 3 minutes at 12,000 rpm on a Beckman microcentrifuge (USA). The supernatant (1 ml) was transferred to tubes, 1 ml of phosphate buffer and 0.5 ml of hexadecane were added. The contents of the tube are mixed on a mixer for 2 minutes. As control use 0.5 ml of hexadecane in 2 ml of phosphate buffer. The surface activity is assessed by the optical density of the studied samples on a FEK-56M-U42 spectrophotometer at a wavelength of 540 nm and a cell thickness of 0.5 cm. As can be seen from the data in Table. 1, all investigated microorganisms have high emulsifying activity.

Analysis of the results allows us to attribute the majority of bacterial strains to the endotype, i.e. the bioemulsifiers they produce are associated with the cell wall and the optical density of the culture fluid after centrifugation in the presence of hexadecane was low. The bacterial strain Rhodococcus sp. VKM Ac-2533D can be attributed to the exotype, because a bacterial strain releases bioemulsifiers into the culture fluid. As can be seen from the above data, the presence of 3% NaCl in the medium leads to a decrease in the emulsifying activity exhibited by microorganisms.

Thus, an association of four psychrotrophic microorganisms producing bioemulsifiers and capable of degrading oil and oil products at high salt concentrations both at normal and at low temperatures has been proposed.

Claims (1)

Association of bacteria strains Rhodococcus sp. BKM Ac-2532D, Rhodococcus sp. BKM Ac-2533 D, Pseudomonas sp. BKM B-2387D and Pseudomonas putida BKM B-2380D, producing bioemulsifiers, for the degradation of oil and oil products in soils, fresh and sea water.