RU2673747C1 - GORDONIA AMICALIS STRAIN WITH ABILITY OF GENERATION DIRECTLY IN OIL RESERVOIR OF OIL-DISPLACING AGENT - bioPAV AND DECREASING CONTENT OF ORGANOSULFUR COMPOUNDS OF OIL - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of Gordonia amicalis 6-1, capable of being generated directly in the oil reservoir of an oil-displacing agent – bioPAV and reducing the content of organic sulfur compounds in oil, deposited in the All-Russian Collection of Microorganisms of Skryabin Institute of Biochemistry and Physiology of Microorganisms (IBPM RAS) under the VKM registration number of Ac-2795D. Strain of Gordonia amicalis VKM as Ac-2795D can be used in microbial methods to improve oil recovery.

EFFECT: invention allows to increase the degree of oil recovery.

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Description

The strain Gordonia amicalis, capable of generating directly in the oil reservoir an oil-displacing agent - bio-surfactant and reducing the content of organosulfur compounds in oil.

The invention relates to the oil industry, and can be used in microbial methods to increase oil recovery using bacterial strains. It is known that different types of microorganisms are widely used in oil production technologies. For example, in the patent of the Russian Federation 2539483 it is proposed to pump a composition including microorganisms Pseudomonas sp. In order to increase oil recovery. and Rhodoccocus erythropolis. In patent 2539485 it is proposed to use for this purpose the biological product Devoroil, which includes representatives of the genera Pseudomonas, Candida and Rhodococcus. The disadvantage of these compositions is that 50 g / L of strong alkali (NaOH) is added to the injected solution along with microorganisms, which leads to an increase in the pH of the solution to pH≈13, which is incompatible with the vital activity of the injected microorganisms.

US patent 7,708,065 proposes the use of a Thauera AL9: 8 strain, US 7,776,795 a Shewanella putrefaciens LH4: 18 strain and US 8,357,526 Pseudomonas stutzeri to improve oil recovery under anaerobic formation conditions. It is believed that the development of microorganisms in the reservoir will occur due to the oxidation of oil and the reduction of nitrates in the process of denitrification. The disadvantage of these patents is that nitrates must be pumped into the reservoir at the same time and the process of oil oxidation under anaerobic conditions proceeds at a very low speed, i.e. profitability and efficiency of use of these strains is low.

In RF patents 2125152 and 2307148, in which bio-surfactants isolated by the Pseudomonas aeruginosa S-7 strain are used as an oil displacing agent during cultivation in a fermenter. The disadvantage of this approach is that the oil displacing agent is produced in the factory, which significantly reduces the cost-effectiveness of using the strain. In addition, P. aeruginosa belongs to opportunistic microorganisms, which leads to additional costs associated with the safety of the fermentation process and the subsequent manufacture of a commodity form of oil-displacing agent.

For microorganisms belonging to the genus Gordonia, properties are unknown that allow their use in oil recovery technologies.

RF patent 2477316 describes the ability of the bacterial culture of Gordonia terrae IEGM136 (VKPM AC-1897) to synthesize (R) -phenylmethyl sulfoxide by transformation of phenyl methyl sulfide.

The technical task of the invention is the selection of a strain capable of generating directly in the oil reservoir oil-displacing agent and reducing the content of organosulfur compounds in oil.

EFFECT: improved oil recovery under anaerobic formation conditions and a decrease in the content of organic sulfur compounds of oil.

It is achieved by the fact that the inventive Gordonia amicalis 6-1 is proposed as a means for generating an oil-displacing agent, bio-surfactant directly in the oil reservoir, and which reduces the content of organosulfur compounds in oil.

Strain Gordonia amicalis 6-1 was isolated from a prefabricated oilfield pumped into the Cheremukhovskoye oil field, Republic of Tatarstan. The strain is deposited in the All-Russian collection of microorganisms (VKM Ac-2795D).

Characterization of the strain Gordonia amicalis 6-1.

The strain is represented by gram-positive small sticks. On agar medium PCA (bacto-tryptone - 5 g / l, yeast extract - 2.5 g / l, glucose - 1 g / l, NaCl - 5 g / l, microbiological agar - 15 g / l) forms small round mucous membranes colony of orange color. The strain grows aerobically in the presence of from 0 to 100 g of NaCl / l of medium (optimum 15 g / l); in the pH range from 6.5 to 8.2 (optimum 7.4); at a temperature of 5 to 45 ° C (optimum 28 ° C). Strain 6-1 was identified by analysis of the 16S rRNA gene as a representative of the species Gordonia amicalis (the sequence was deposited at Genbank under the number AN MN101280). It uses as a sole source of carbon and energy sugar (sucrose, glucose, arabinose), amino acids (alanine, glutamic acid), alcohols (methanol, ethanol, glycerin), sodium salts of acetic, lactic and propionic acids, as well as individual hydrocarbons and crude oil . The strain uses n-alkanes of oil and organosulfur aromatic compounds, produces surfactants (bio-surfactants), which reduce the interfacial tension at the interface of the aqueous and hydrocarbon phases to 0.1-1.0 mN / m. The strain is stored on beveled PCA agar and in a lyophilized state at a temperature of + 4 ° C.

To obtain cell biomass, the strain is expanded in a liquid nutrient medium of the following composition, g / l: NH ₄ NO ₃ - 0.5, K ₂ HPO ₄ - 1.5, KH ₂ PO ₄ - 0.75, CaCl ₂ ⋅ 2H ₂ O - 0.02, MgSO ₄ ⋅ 7H ₂ O - 0.2, KCl - 0.1, NaCl - 5.0; tap water, pH - 7.0 -7.2. Petroleum hydrocarbons or soluble substrates are used as the sole carbon source, making it easier to separate microbial biomass. Cultivate at a temperature of 25-30 ° C under aerobic conditions until the titer of microorganisms in the culture fluid reaches at least 10 ⁸ CFU / ml.

The oil-displacing ability of the hydrocarbon-oxidizing strain G. amicalis 6-1 is supposed to be used during its injection and activation directly in the oil reservoir. In this case, the residual oil in the bottomhole zone of the injection well will be used as an organic substrate. The composition of the cell wall of G. amicalis 6-1 includes mycolic acids, which give the strain oleophilic properties, which contributes to its concentration at the border of the oil-water contact. The selection of bio surfactants directly in the oil contact zone leads to a significant decrease in interfacial tension and a decrease in the effect of capillary forces that hold oil in micropores of oil-containing rock. As a result of bio-surfactant exposure, the mobility of oil increases and the degree of its extraction from the reservoir increases.

It is known that the quality and price of oil decrease with an increase in the content of organosulfur compounds in it. Removing the latter during the oil refining process requires additional costs. Therefore, reducing the concentration of organosulfur compounds in oil prior to its refining is an economically important task. The strain G. amicalis 6-1 was found to be capable of desulfurization of crude oil, confirmed by the detection of growth on dibenzothiophene and due to the presence of a specific gene (dszB) in the genome. This gene was detected in strain 6-1 by the method of polymerase chain reaction with primers bdsBf (5'-atcgaactcgacgtcctcag-3 '), bdsBr (5'-ggaacatcgacaccaggact-3') specific for the dszB gene.

The described properties of the strain G. amicalis 6-1 are illustrated by the following examples.

Example 1. The study of the formation of bio-surfactants and reduce surface and interfacial tension strain Gordonia amicalis 6-1 when cultured in a medium with soluble substrates (glucose).

The strain Gordonia amicalis 6-1 was grown on a medium of the following composition: CaCl ₂ × 6H ₂ O - 0.01; MnSO ₄ × 5H ₂ O - 0.02; FeSO ₄ × 7H ₂ O — 0.01; Na ₂ HPO ₄ - 1.5; KH ₂ PO ₄ - 1.0; MgSO ₄ × 7H ₂ O 0.2; NH ₄ NO ₃ - 1.0; NaCl - 5.0; glycerin (or glucose) - 20.0; yeast extract - 0.5; distilled water to 1000 ml, pH 6.8-7.2 (adjusted with 0.1 M HCl or NaOH). The medium is poured into 100 cm ³ into a rocking flask with a volume of 500 cm ³ , closed with a cotton plug and sterilized in an autoclave. Sterilization mode - flowing steam, temperature 121 ° C for 20 minutes.

After cooling the mineral medium to room temperature, Gordonia amicalis 6-1 culture was sterilized in the flasks with a final titer of about 10 ⁶ CFU / ml of medium. After 2, 5, and 10 days of cultivation on a shaker (120 rpm) at a temperature of 30 ° C, the surface (PN) and interfacial (MN) tension of the culture medium was measured. The magnitudes of PN and MN were measured by the semistatic method of ring detachment on a semi-automatic tensiometer Surface Tensiomat 21 (Cole-Parmer, USA) equipped with a platinum-iridium ring. The method of separation of the ring is to determine the separation force of the ring or loop of wire from the surface of the liquid. This method is one of the variants of the Wilhelmy plate method. MN was measured at the phase boundary: culture fluid / hexadecane. Samples were kept for 30 min to form a phase boundary. The measurements were carried out at a temperature of 25 ° C.

The research results are shown in table 1, show that the culture fluid with cells of the strain Gordonia amicalis 6-1 after 5 days of cultivation had high oil-displacing properties. The surface tension of the culture fluid of strain G. amicalis 6-1 was 16 mN / m (prototype 35 mN / m), and the interfacial tension (against hexadecane) was 0.1 mN / m (prototype 0.5 mN / m )

Example 2. The study of the formation of bio-surfactants and reduce surface and interfacial tension by strain Gordonia amicalis 6-1 when cultivated in a medium with crude oil as the sole carbon source.

The strain Gordonia amicalis 6-1 was grown on a medium of the following composition: CaCl ₂ × 6H ₂ O - 0.01; MnSO ₄ × 5H ₂ O - 0.02; FeSO ₄ × 7H ₂ O — 0.01; Na ₂ HPO ₄ - 1.5; KH ₂ PO ₄ - 1.0; MgSO ₄ × 7H ₂ O 0.2; NH ₄ NO ₃ - 1.0; NaCl (0.5%) - 5.0; distilled water to 1000 ml, pH 6.8-7.2 (adjusted with 0.1 M HCl or NaOH). After cooling the sterile mineral medium to room temperature, a G. amicalis 6-1 culture with a final titer of about 10 ⁶ CFU / ml of medium is sterilized in the flasks. Then, 5 cm ^{3 of} crude sterile oil of the Romashkinskoye field (Devonian horizon) is added to the flask, closed with a cotton plug and placed on a rocking chair (120 rpm) at a temperature of 30 ° C. For control, a flask with medium and oil is placed on a rocking chair without introducing a bacterial strain into it. After 7 days of incubation, the flask was removed from the rocking chair and in a culture medium containing biomass and emulsified oil, the pH, MN at the border with hexadecane were measured. In addition, the culture medium was centrifuged and the supernatant was separated from the biomass and residual oil. Similar measurements are performed in the supernatant.

The research results shown in table 2 show that the medium containing biomass and emulsified oil, after 7 days of cultivation, had the same low surface tension values as in option 1, where glucose was used as an organic substrate. It was shown that the supernatant separated from the cells can also be used as an oil displacing agent, but its effectiveness in this capacity is lower.

Example 3. The study of the ability of strain G. amicalis 6-1 to wash oil from the surface of the mineral matrix.

Quartz sand and carbonate sand were used as a mineral matrix. Heated high-viscosity oil of the Cheryomushkinskoye field was added to the sand samples to complete saturation. The sample was weighed and transferred to a flask with a mineral medium of the following composition: CaCl ₂ × 6H ₂ O - 0.01; MnSO ₄ × 5H ₂ O - 0.02; FeSO ₄ × 7H ₂ O — 0.01; Na ₂ HPO ₄ - 1.5; KH ₂ PO ₄ - 1.0; MgSO ₄ × 7H ₂ O 0.2; NH ₄ NO ₃ - 1.0; NaCl - 5.0; distilled water to 1000 ml, pH 6.8-7.2 (adjusted with 0.1 M HCl or NaOH). Then, Gordonia amicalis 6-1 biomass was introduced into the flasks and incubated on a shaker (120 rpm) at a temperature of 30 ° C. G. amicalis 6-1 was not added to the control flasks. After 15 days of cultivation, sand samples were separated from the liquid phase, washed twice with water, dried in an oven and brought to constant weight. The ability of G. amicalis 6-1 to wash oil from the surface of the mineral matrix was judged by the difference in weight of sand samples before and after the experiment.

The research results are shown in table 3, show a high degree of washing of oil from the surface of mineral particles with a culture medium with bio-surfactant strain G. amicalis 6-1. In the control, about 88% of the oil remained bound to the sand surface. In the experiment with G. amicalis 6-1, this value did not exceed 24% for quartz and 43% for carbonate material.

Example 4. The study of the ability of strain Gordonia amicalis 6-1 to reduce the content of organosulfur compounds in oil.

The strain Gordonia amicalis 6-1 is grown on a medium of the following composition: CaCl ₂ × 6H ₂ O - 0.01; MnSO ₄ × 5H ₂ O - 0.02; FeSO ₄ × 7H ₂ O — 0.01; Na ₂ HPO ₄ - 1.5; KH ₂ PO ₄ - 1.0; MgSO ₄ × 7H ₂ O 0.2; NH ₄ N0 ₃ - 1.0; NaCl (0.5%) - 5.0; distilled water to 1000 ml, pH 6.8-7.2 (adjusted with 0.1 M HCl or NaOH). The medium is poured into 100 cm ³ into rocking flasks with a volume of 500 cm ³ , and closed with a cotton plug. After cooling the sterile mineral medium to room temperature, G. amicalis 6-1 culture with a final titer of about 10 ⁶ CFU / ml of medium was sterilely introduced into the flasks. Then, 5 cm ^{3 of} crude sterile oil of the Romashkinskoye field (Devonian horizon) enriched with dibenzothiophene (DBT) in an amount of 500 mg / l of medium was added to the flask, closed with a Teflon stopper to prevent loss of dibenzothiophene and placed on a rocking chair (120 rpm). Incubated at a temperature of 30 ° C. For control, a flask with medium and oil with benzothiophene was incubated on a rocking chair without introducing a bacterial strain into it. Every 3 days, air was introduced into the flasks.

After 15 days, the flasks were removed from the rocking chair and oil was separated from the culture medium in a separatory funnel. After cultivation with G. amicalis 6-1, the dibenzothiophene (DBT) content in the medium decreased from 500 mg / L (3.0 mmol) to 12 mg / L (0.06 mmol), thus 98% DBT was consumed. In the control, the dibenzothiophene content did not change. Strain G. amicalis 6-1 used dibenzothiophene as a sulfur source only in the presence of other organic sources of carbon and energy.

Claims (1)

The strain Gordonia amicalis, deposited in VKM under the number Ac-2795D, capable of generating directly in the oil reservoir an oil-displacing agent - bio-surfactant and reducing the content of organosulfur compounds in oil.