RU2103359C1 - Strain of bacterium erwinia species vkpm b-7005 - a destructor of petroleum products and organic substrate in plant raw and animal husbandry waste - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: new strain of bacterium Erwinia species VKPM B-7005 isolated from natural medium (oil-bearing, core) is selected in processes of manure and dung processing under anaerobic and aerobic conditions. This strain decomposes organic substrates in plant raw and animal husbandry waste under aerobic and anaerobic conditions, oil and petroleum products in the process of treatment of different objects from these substances and hydrocarbon substrate at evolution of large amount of gaseous products at high rate. EFFECT: strain indicated above, enhanced effectiveness of strain.

Description

 The invention relates to the field of microbiology and can be used in the oil industry to increase oil recovery of oil reservoirs by microbiological methods, biotechnology, agriculture for the decomposition of organic substrates in plant materials and animal waste, for example, for aerobic and anaerobic processing of manure, poultry manure, in any areas human activities for cleaning objects from petroleum products.

 Known strains of microorganisms used to increase oil recovery of oil reservoirs by microbiological methods.

 For example, a method is known [1] for injecting into a formation a suspension of a culture of bacteria p.Clostridium. A mixture of adapted microorganisms is introduced into the reservoir, then a 2.5% molasses solution with polyphosphate salts and soda, which are dissolved in a mixture of fresh and produced waters in a ratio of 1:10. At the same time, core models show an increase in oil recovery by 16.5 - 47.5%, and an increase in oil production at the well by 3 times (from 50 to 150 tons / day) with a decrease in the degree of water cut from 80 to 60%.

 The disadvantage of this method is that the indicators of the oil production process described in the description of this method are not mutually linked. The increase in oil production in the well is three times, and the increase in oil recovery in core models is only 16.5-47.5%, which indicates a very insignificant effect of this method. It can be assumed that the increase in oil production at the well was achieved only in a short initial period after the application of the method.

 Known strains of microorganisms - destructors of petroleum products used to clean any objects from oil and its products.

 For example, in [2], the bacterial strain Pseudomonas putida - 36 is used for water and soil purification, which allows utilization of up to 75% of oil when the oil content to the volume of contaminated substrate (soil, water) is up to 10%. The disadvantage of this method is the cleaning of only slightly contaminated objects.

 Known strains of microorganisms used in biotechnology, agriculture for the decomposition of organic substrates in plant materials and animal waste.

For example, in [3], thermophilic bacteria of the genus Lactouticterium or Streptouacterium mesophilic bacteria of the genera Streptococcus, Azotobacterium, Pseudomonas are used to decompose manure on a medium containing also carbon sources, mineral acids and proteins, at temperatures of 5-35 ^o C for 7-14 days. The disadvantage of this method is the processing of highly diluted manure for a long period of time.

 The technical problem solved by the present invention was to isolate from the natural environment (oil-bearing rock, core, the Khadyzhenskoye field of the Grozny oil-producing enterprise (NDP)) and selection in the processing of manure, poultry manure in anaerobic and aerobic conditions, decomposition of oil and oil products during refining objects from petroleum products, in model experiments to increase oil recovery of oil-bearing rocks of a strain capable of decomposing organic substrates in plant materials with high speed and from odes animal under aerobic and anaerobic conditions, oil and petroleum products from the cleaning process various objects from these substances, the hydrocarbon substrate with the release of large quantities of gaseous products.

 The technical result obtained as a result of the implementation of the invention consists in the ability to carry out with great speed and high yield the processes of destruction of oil and oil products at facilities to be cleaned of these products, the decomposition of the organic substrate in plant materials and animal waste to achieve a stable high result with increased oil production oil reservoirs, and not only by the method of watering the reservoirs with a suspension of microorganisms, but also by the method of cleaning oil wells.

The proposed new strain of microorganisms Erwinia species 206-B 1 was obtained by sequentially isolating it from an association of microorganisms obtained by culturing them under periodic aerobic and anaerobic conditions (B) for 2 days at a temperature of 32 ^o C on a medium of the following composition (A ), g / l:
oil rock (core) - 10: glucose - 10: oil 10; medium M 9 with m / s "P" of the Institute "GosNIIsintezbelok" Isolation of a new strain of bacteria was carried out by repeatedly plating the association of the microorganism obtained on the above liquid medium during cultivation, on solid media (wort agar and meat peptone agar), separation of colonies of microorganisms , further culturing the colonies in the above liquid medium (A) under the above conditions (B) and subsequent plating on solid media, etc.

 Determination of the systematic position of the selected strain, carried out in accordance with Bergie’s “Bacterial Identifier” (Bergy'S Vfnual of Systematic Bacteriology-Baltimore: Hong Kong: London. Sidney - 1984, 1986, 1989. v. 1.2.3.4.) Showed its belonging to Erwinia species .

 Determination of the degree of pathogenicity of the isolated and selected strain of Erwinia species 206-B1 confirmed the absence of pathogenicity. Characterization of the bacterial strain Erwinia species 206 B 1.

The composition of the medium N 9 with m / e "P", g / l: NH ₄ • H ₂ PO ₄ - 10; K ₂ HPO ₄ • 10; MgSO ₄ • 7H ₂ O - 0.7; NaCl - 10; mg / l: F ₂ So ₄ - 15; MnSO ₄ - 15; ZnSO ₄ -15; pH = 6.7-7.0
Cultural morphological characters.

 Erwinia spesies 206 - B1 forms colonies on meat-peptone agar with glucose and a 1-3 mm round yeast extract with a flat or scalloped edge, shiny to matte, convex or flat, from colorless at a young age to tan. The cells are short, (0.7-1.0 x 1.0-2.5 microns), motile in the first hours of growth of the stick.

 Physiological biochemical signs.

The strain Erwinia spesies 206 - B1 with a periodic method of cultivation under aerobic conditions at a temperature of 32-36 ^o C and pH = 5-7 completely utilizes petroleum products for 36-48 hours

 It decomposes the organic substrate in plant materials, for example, it is suitable for aerobic and anaerobic treatment of manure and manure.

 It synthesizes low molecular weight acids, aldehydes, alcohols on a carbon substrate.

It is stored in test tubes at 4 ^o C under oil on meat-peptone agar with 2% glucose and 0.1% corn extract, pH = 7.0.

Propagated in test tubes at 30 ^o C on meat-peptone agar with 2% glucose and 0.1% corn extract, pH = 7.

 The isolated new strain of microorganisms was selected in the processes of processing manure, bird droppings under anaerobic and aerobic conditions, decomposition of oil and oil products when cleaning objects from oil products, in model experiments to increase oil recovery.

The process of processing manure and bird droppings was carried out in a laboratory unit with mechanical stirring of the processed material and (under aerobic conditions) air supply to the system. The processed material (manure or litter) was loaded into the apparatus, neutralized with phosphoric acid solution to pH = 7.0, sterilized for 1 h at a temperature of 130 ^o C and 1 atm, and under sterile conditions introduced into the apparatus 1% of the mass. by weight of the processed material culture of the new strain. The processing of the material was carried out at a temperature of 32 ^o C for 48 h, after which the processed material in an amount of 1% of the mass. loaded into the nutrient medium of the above composition (A), the strain was cultured under the above conditions (B), plated on solid media, tested for monoculture, and in the next experiment, the apparatus was seeded with a strain culture grown on liquid medium A. The experiments were repeated in this way until until the complete necessary processing of manure or litter was achieved in 48 hours of the process.

The process of decomposition of oil and oil products was carried out during the cleaning of soil samples (loam) taken near production wells from different depths of the soil of the Aktobe NDP polluted to varying degrees with oil. Samples were loaded into the apparatus, wetted with solutions of ammonium sulfate and monosubstituted potassium phosphate so that the concentrations of nitrogen, phosphorus and potassium in the samples were, respectively, equal to: 1.0; 0.6; 0.2 g / g of soil. Sterilized for 1 h at a temperature of 130 ^o C and 1 ATM and in sterile conditions was introduced into the apparatus 1% of the mass. by weight of the processed material culture of the new strain. The processing of the material was carried out at a temperature of 34-36 ^o C for 7 days at a constant moisture content of the material (75%) and a constant concentration of nitrogen, phosphorus and potassium with mechanical stirring of the material, after which the processed material in an amount of 1% of the mass. loaded into the nutrient medium of the above composition (A), the strain was cultured under the above conditions (B), plated on solid media, tested for monoculture, and in the next experiment, the apparatus was seeded with a strain culture grown on liquid medium A. The experiments were repeated in this way until until the cleaning of samples at the level of 80-90% of the initial pollution was achieved in 7 days of the process.

Model experiments to increase oil recovery of oil-bearing rock were carried out as follows. In a special apparatus, a tube simulating a production well was treated with heavy oil fractions (paraffinic and resinous-asphaltene) so that the pore volume in the rock filling the tube was reduced to 10% of the total volume. With the initial physical parameters in the apparatus simulating the oil reservoir: temperature - 45 ^o C and pressure -10 MPa, the oil extraction from the tube was 9 ml / min. Then, on a mixture of tap and produced water taken in a 1: 1 ratio, a 1% suspension of a new strain was prepared, to which was added, g / l: corn stalk hydrolyzate with a sugar content of 40% - 120.0; ammonium nitrate - 2.0; monosubstituted potassium phosphate - 1.0; magnesium sulphate heptahydrate - 0.2. The prepared biologically active medium is pumped into the rock of a tube simulating a production well and kept there for 360 hours. (All operations and the entire process were carried out under sterile conditions) Then the tube was opened and oil sampling was measured. Then the rock from the tube in an amount of 1% of the mass. loaded into the nutrient medium of the above composition (A), the strain was cultured under the above conditions (B), plated on solid media, tested for monoculture, and in the next experiment, the apparatus was seeded with a strain culture grown on liquid medium A. The experiments were repeated in this way until until the necessary cleaning of the rock in the tube and the necessary increase in the pore volume were achieved in 360 hours of the process, and, therefore, the necessary increase in oil extraction from the tube was not obtained.

The invention is illustrated by the following examples.
Example 1. In a special apparatus simulating oil production from an oil reservoir, a tube simulating a production well was treated with heavy oil fractions (paraffinic and tar-asphaltene) so that the pore volume in the rock filling the tube was reduced to 10% of the initial volume. At the initial physical parameters in the formation: temperature -35 ^o C and pressure -10 MPa, oil extraction from tube 1 under these conditions was 9 ml / min. A working suspension of bacteria p.Erwinia spesies VKPM B-7005 was prepared. To do this, the biomass of bacteria was accumulated in the fermenter at a temperature of 32 ^o C and a pH of 7.0 in a nutrient medium of the following composition, g / l: oil - 40.0; molasses - 10.0; sodium chloride - 20.0; phosphoric acid - 3.0; ammonium hydroxide - 4.0; potassium chloride 0.8; magnesium sulphate heptahydrate - 0.3 ;, trace elements. After biomass accumulation, it was separated from the liquid phase by centrifugation, preserved and then diluted in a mixture of tap and produced water, taken in a 1: 1 ratio. To the resulting working suspension was added, g / l: corn stalk hydrolyzate with a sugar content of 40% - 120.0; ammonium nitrate - 2.0; monosubstituted potassium phosphate - 1.0; magnesium sulphate heptahydrate - 0.2. The prepared biologically active medium was pumped into the rock of the tube and held there for 360 hours. Then the tube was opened and oil extraction was measured, which was equal to 24 ml / min., I.e. the effect of applying the method in this case was 167%.

 Example 2. The area contaminated with oil on 20% of the soil, for example, loam was treated with a suspension of bacteria Erwinia spesies VKPM B-7005. A suspension of microorganisms was prepared as in example 1, only microorganisms were diluted with river water in an amount of 5 g / l, and ammonium nitrate was also added thereto at a rate of 1.5 g / l, superphosphate at a rate of 1.0 g / l, potassium chloride from calculation of 0.2 g / l and magnesium chloride at the rate of 0.05 g / l (sources of nitrogen, phosphorus and potassium can be replaced with an equivalent amount of complex fertilizer - ammofoski). The soil was watered with a suspension of microorganisms in an amount of 1 liter of suspension per 1 m of soil, and then loosened. For 15 days, the soil was watered either with water or with a solution of the above salts (depending on the current concentration of nitrogen and phosphorus in the soil) and loosened. After 15 days, the degree of soil contamination was determined equal to 2.7%.

Example 3. The process of processing cattle manure was carried out in a laboratory setting with mechanical stirring of the processed material and air supply to the system. The processed material was loaded into the apparatus, neutralized with a solution of phosphoric acid to pH = 7.0 and introduced into the apparatus I% wt. by weight of the processed material culture Erwinia spesies. The processing of the material was carried out at a temperature of 34-36 ^o C for 48 hours and subsequent drying at a temperature of 80 ^o C for 5 hours. Received a material with the following characteristics: pH - 6.5 - 7.5; content,%: moisture - 14; nitrogen (total) - 5.0; nitrogen (ammonium) -0.7; phosphorus - 0.5; potassium - 1.5; carbon (total) - 28; including humic acids - 6.0; BOD ₅ - 550 units; pathogenic microorganisms are absent; helminth eggs and larvae (viable) - absent; sharp specific smell - absent; good flowability, absence of large lumps (no more than 5 mm).

Literature:
1. Microbial enhancement of oil recovery from carbonate reser -
voirs with complex formation characteristics / Wagner M. // Microbial Enhanc. Oil Recovery Recent Adv .: Proc. Int. Conf., Norman, Okla, 1990 - Amsterdam, 1991.

 2. A. S. USSR N 1076446. The strain Pseudomonas putida-36, used to purify water and soil from oil products.

 3. British patent N 2026087. Food for animals obtained by the microbiological method.

Claims (1)

 The bacterial strain Erwinia species VKPM B-7005 is a destructor of oil products and organic substrate in plant materials and animal waste.