RU2129604C1 - Consortium of microorganisms arthrobacter oxidants and pseudomonas putida used for treatment of soil and water from petroleum and petroleum products pollution - Google Patents

Abstract

FIELD: technical microbiology, ecology. SUBSTANCE: consortium of microorganisms Arthrobacter OXIDANTS Ac-1838 and Pseudomonas putida B-2443 are used for treatment of water and soil from pollution with petroleum and petroleum products. Consortium is able to utilize petroleum hydrocarbons at the broad range of pH medium. Degradation degree of biochemical oxidation at pH 8.9 for 96 h is 87.4%. EFFECT: high degradative capability of consortium. 5 tbl, 5 ex

Description

 Usage: purification of soils contaminated with oil and oil products, fresh and salt water and settling ponds in various climatic conditions.

 The inventive created a consortium of microorganisms Arthrobacter oxydans Ac-1838D, Pseudomonas putida B-2443, which provides effective treatment of water and soil from contamination by oil and oil products.

 Known biopreparations created on the basis of monocultures are: Putidoyl (author's certificate N 1076446, 1982, author certificate N 1428809, 1988); Bioprin (Oleovorin) TU 11279895-12-22-92 ed. testimonial. N 1809822, 1993, autosvid. N 2014286, 1994) used to clean water and soil from oil pollution. Each of these drugs is equally inherent in certain advantages, which were further supplemented and expanded.

Methods are proposed for operation at low temperatures of 8-10 ^° C. (autoswitch. N 2019527, 1994) and high temperatures (autoswitch. N 2014286).

 A known method of cleaning soil and water from oil pollution at low pH values of 3.5-6.0 (ed. Certificate. N 2007372, 1994).

 Consortia of microorganisms are proposed for wastewater treatment from phenol, methanol and formaldehyde (author certificate No. 1387343, 1986), from diethylene glycol (author certificate No. 1774602, 1990), from ethylene glycol and diethanolamine (author certificate No. 1800800, 1990 )

 The closest to the proposed is a consortium of microorganisms "Devoroil" (autoswitch. N 2023686, 1994), actively oxidizing oil products both in the contact zone with water and directly in the oil film, capable of utilizing a wide range of oil hydrocarbons both in fresh and in in a saline ecosystem.

 The disadvantage of this consortium is the complexity of the technology for preparing the drug from five cultures.

 The purpose of the invention: the creation of a consortium of microorganisms capable of utilizing oil and oil products in the soil, in fresh and sea water and for the treatment of settling ponds with alkaline pH (8-9).

 The proposed goal is achieved by using a consortium of microorganisms in combination with mineral additives of nitrogen (ammonium salt) and phosphorus.

As associative microorganisms, G ⁺ (gram-positive) - Arthrobacter oxydans Ac-1838D and G ^- (gram-negative) - Pseudomonas putida B-2443 bacteria were used.

 The advantage of the proposed consortium is the combination of two strains in one association that exhibit unequal activity with respect to the nutrient substrate (Arthrobacter oxydans oxidizes aromatic oil more efficiently, and Pseudomonas putida paraffinic) and environmental conditions (Ps. Putida oxidizes hydrocarbons more efficiently at slightly acidic and neutral values pH, a Arthrobacter oxydans - in neutral and alkaline conditions).

 The composition allows you to make the process of biodegradation of oil and its derivatives more effective (degree of purification above 95% for 96 hours of destruction).

 The main feature of the consortium is that it proposes a new strain of Arthrobacter oxydans Ac-1838D in association with Pseudomonas putida B-2443, growing at pH above 8 and actively degrading oil and oil products.

The strain Arthrobacker oxydans Ac-1838D was isolated from soil samples taken from the oil fields of the Astrakhan OGPD (Kalmykia) in 1991. The strain obtained by the method of cumulative culture, i.e. repeated reseeding on a liquid mineral medium with oil. For cultivation, Voroshilova-Dianova medium was used with the following composition (g / l):
K ₂ HPO ₄ - 1.0; KH ₂ PO ₄ - 1.0; NH ₄ NO ₃ - 1.0; MgSO ₄ 0.2; CaCl ₂ • 6 H ₂ - 0.01; FeCl ₃ • 6 H ₂ O - 3 drops in the presence of 1% hydrocarbons.

 Cultivation was carried out in flasks on a rocking chair 180 rpm, the volume of the medium 200 ml, the oil concentration of 1%.

 The proposed strain is characterized by the following morphological-cultural and physiological-biochemical properties.

 1. Morphological and cultural characters.

 Kokk - a stick, - a coccal growth cycle. It does not form endospores. Gram-positive, motionless, does not ferment. Metabolism is respiratory, never fermentative. On the IPA, it forms creamy-white colonies that are round, convex, smooth, shiny, rastiform.

 2. Physiological and biochemical characteristics.

Aerobe. The optimum growth is 20-30 ^o C. It grows at a temperature of 10-45 in the range of pH 5.5-9, salinity up to 6% NaCl.

 Attitude to carbohydrates.

 Uses sucrose, lactose, maltose, galactose, glucose, raffinose, mannitol, glycerin, does not absorb arabinose.

 Attitude to hydrocarbons.

 Uses octane, hexane, toluene, xylene, benzene, paraffin, asphaltenes, camphor. Does not absorb naphthalene. Aromatic oils are absorbed more actively than paraffin oils.

 Attitude to power sources.

 Assimilates nitrate nitrogen.

 Other characteristic physiological features of metabolism.

 Gelatin does not dilute, starch does not hydrolyze, and does not form hydrogen sulfide and indole. Ammonia does not emit. The oxidase reaction is negative, catalase-positive. It does not have lecithinase, arginine dehydrolase, urease, lysine decarboxylase.

 Information about the presence of pathogenic properties.

 Non-pathogenic.

 Table 1 presents data on the effect of various antibiotics on the Arthrobacter oxydans strain.

 Based on the complex of morphological, cultural, physiological and biochemical properties, the strain was identified as Arthrobacter oxydans and deposited in the All-Union Collection of Microorganisms IBPM under registration number VKPM AC-1838D.

 Storage conditions.

Sterile columns (MGTA medium) are incubated at 30 ^o C for 48-72 hours, pour 1 ml of sterile paraffin oil and stored at 4 ^o C, reseeded 3 times a year.

Strain Pseudomonas pukida B-2443
Isolated from organic-based drilling mud (oil) stored in an open-cut sludge pit of the Belozersky deposit in the Tyumen Region and deposited at VNIIGenetika, collection number TsMPM V-2443.

 1. Morphological and cultural characters.

 The size of the cells of a one-day culture is 1.7-2.4 x 0.3-0.5 microns. Gram-negative. The predominant form of cells is straight, rod-shaped. On the MPA, colonies form colorless, convex, round, 3-7 mm in size. On the meat and peptone broth form turbidity with the release of gas. On potatoes - abundant growth, a convex stroke, continuous with the formation of mucus. It does not dilute gelatin. Milk coagulates, acidifies.

 2. Physiological signs.

Aerob grows at 28-40 ^o C, at 4 ^o C does not grow.

 Attitude to carbohydrates.

 Uses lactose, glucose, and maltose with evolution of gas. Does not absorb arabinose.

 Attitude to hydrocarbons.

 Uses naphthalene, camphor, octane, hexane, benzene, toluene, xylene, paraffin, asphaltenes. Paraffinic oils assimilate more actively than aromatic oils.

 Attitude to power sources.

 Assimilates nitrate nitrogen.

 3. Storage of the strain.

The strain is stored in the collection in a lyophilized state. To maintain the strain using the method of periodic reseeding (1-2 times a year) under a layer of paraffin oil at a temperature of 4 ^o C.

Example 1. Periodic cultivation of the association of cultures of Ps.putida and A. oxydans was carried out at 30 ^o C on a shaker with 180 rpm The culture medium is given above. The stability of the association is confirmed by seeding on a solid nutrient medium with the counting of colonies of various types.

Table 2 presents the results of a study of the destructive activity of individual strains of the association and the bacterial association itself, taken in equal proportions. The concentration of microorganisms is 10 ⁹ cells per 1 ml of medium. Contact time 96 hours. The change in the oil content in the culture medium was controlled by gas-liquid chromatography on a Chrom-5 chromatograph.

 As can be seen from table 2, the association under periodic conditions has a greater destructive activity compared to individual strains. Paraffin oil is utilized more efficiently (95.8%) than aromatic oil (92.4%).

Example 2. The study of the process of utilization of hydrocarbons from prolonged exposure
The experiment was carried out by analogy with example 1.

 Table 3 presents the results of biodegradation of oil and diesel fuel depending on the exposure time of the experiment.

 From table 3 it is seen that the maximum degree of decomposition of diesel fuel is achieved on the fourth day, in contrast to the analogue, where the maximum degree of destruction of diesel fuel occurs on the 5th day.

 The maximum degree of oil destruction (> 95%) in experiments with the association of bacteria also occurs on the fourth day, unlike the analogue, where the percentage of utilization on the fourth day reaches only 75%.

Example 3. Microbial destruction of oil was carried out on a medium Voroshilova-Dianova at a temperature of 28 ^o C. The pH of the medium in the experiments ranged from 3 to 9, the pH was regulated with a 10% solution of NaOH.

 Inoculated one-day material was grown on MPA, washed with sterile saline and introduced into the flasks 5 vol.%, Hydrocarbons were added in an amount of 2 vol.%.

 The results of the effect of pH on the decomposition of oil are presented in table 4.

 The data in the table show that for 96 hours of destruction at pH 8.9, up to 87.4% of the oil is utilized by the association, during the same time only 13.2% is utilized by the analogue.

 Example 4. There was a soil plot of 1 ha located on the territory of NGDU Chernogorneft (Nizhnevartovsk), contaminated with crude aromatic Nizhnevartovsk oil with an intensity of pollution of 7540 mg / 100 g of soil.

The treatment was carried out with an aqueous solution containing nitrogen-phosphorus additives (0.07%) and biomass in the association of Ps.putida and A.oxydans in the form of a concentrated paste (cell titer in a solution of 2 • 10 ⁸ cells / ml. Solution consumption 1 m ³ / ha.

 Before the introduction of the drug, the plot was loosened. Envelope sampling was performed on days 14, 21, 35, 42.

 As can be seen from table 5, the degree of oil utilization on day 42 was 97.9%. Intensive germination of the grass cover in the treated area was observed.

 Example 5

There was a water area of 3 hectares located on the territory of the NGDU Chernogorneft (Nizhnevartovsk) with contamination of crude oil at a concentration of 29.8 kg / m ³ .

The treatment was carried out with an aqueous solution containing nitrogen-phosphorus additives (0.07%) and the biomass of the association of Ps.putida and A.oxydans in the form of a concentrated paste (cell titer in solution of 2 • 10 ⁸ cells / ml). Flow solution of 2 m ^3/10 m ^2.

 A month after the treatment, the oil film disappeared from the surface. The degree of purification is 97%.

 Thus, the proposed consortium can be used to clean the soil and water surface in high pH environments.

Claims (1)

 A consortium of microorganisms Arthrobacter oxydans VKPM Ac-1838D and Pseudomonas putida VKPM B-2443, used to clean the soil and water from pollution by oil and oil products.

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