Halomonas alimentaria for catalyzing rock oxygen consumption in low-temperature oil reservoir air drive and application thereof

Abstract

The application discloses a salt-lactobacillus halophilus for catalyzing rock oxygen consumption in low-temperature oil reservoir air drive, which is preserved in the China General Microbiological Culture Collection Center on September 16, 2022, and the preservation number is CGMCC No. 25748. Meanwhile, the application also discloses application of the salt-lactobacillus halophilus in catalyzing rock oxygen consumption in crude oil exploitation air drive, the strain can use CO2 in air as a carbon source, oxidize S 2‑ ion or elemental S in rock minerals through biological oxidation to obtain energy required for growth, and a large amount of acid is generated in the growth process, further accelerating the dissolution of S 2‑ ion or elemental S and the like in rock minerals, and rapidly consuming oxygen in air, so that the salt-lactobacillus halophilus can be applied as a microbial catalyst in air drive or microbial-assisted air drive oil production, and has good application potential for ensuring air drive safety.

Description

Technical Field

[0001] This invention belongs to the field of oilfield microbial technology, specifically relating to halophilic Lactobacillus in low-temperature reservoirs that catalyzes rock oxygen consumption during air drive and its application in catalyzing rock oxygen consumption. Background Technology

[0002] Air injection is a technology that uses an air compressor to inject air into an oil reservoir, thereby improving oil recovery. It has the advantages of abundant gas sources, low cost, and no geographical or environmental limitations. Previous studies have conducted detailed research on the oxidation principle and oxidation kinetics of air-driven oil recovery, and have concluded that when the reservoir temperature is above 60 ℃, oxygen undergoes a series of complex chemical reactions with crude oil, allowing the oxygen in the reservoir to be consumed within the formation and preventing the danger of oxygen breakthrough.

[0003] Since 2007, air foam flooding tests have been conducted in the Chang 6 reservoir of the Baota Oilfield in the Yanchang Oilfield. The reservoir has a formation temperature of 26-30 °C, a formation pressure of 4.2 MPa, and an average permeability of 0.82 × 10⁻⁶. -3 μm 2 With an average porosity of 7.9%, it is a typical low-temperature, low-pressure, low-porosity, and ultra-low-permeability reservoir. The number of wells has expanded from the initial two to the current 61, proving that air foam flooding is safe and feasible in this reservoir. It was also found that the main factors consuming oxygen during air flooding in low-temperature reservoirs are the adsorption and oxidation reactions of rock minerals, crude oil consumption, and the dissolution reaction of formation water. However, with continuous air injection, the residual oxygen concentration increases, posing a potential safety hazard.

[0004] Microbial oxygen consumption / microbial catalysis of rock mineral oxygen consumption, as a safe and environmentally friendly green technology, has been widely used in various fields such as biometallurgy, biochemical engineering, and environmental protection. In the petroleum industry, microbial oxygen consumption technology is often used for crude oil desulfurization, remediation of oil-contaminated soil, and treatment of oily wastewater. However, research on microbial enhanced oil recovery mainly focuses on the oxygen-consuming activation of endogenous microorganisms or air-assisted microbial oil displacement, while research on microorganisms as biocatalysts for catalyzing rock mineral oxygen consumption has not been reported. Summary of the Invention

[0005] This invention provides a halophilic Lactobacillus strain for air-driven catalysis of rock oxygen consumption in low-temperature oil reservoirs and its application. This strain can utilize CO2 in the air as a carbon source to oxidize sulfur in rock minerals (rich in pyrite) through biological oxidation. 2- Ions or elemental sulfur (S) obtain the energy required for growth, while simultaneously generating large amounts of acid during the growth process, further accelerating the growth of S in rocks and minerals. 2- It may dissolve elemental sulfur (S) and rapidly consume oxygen in the air.

[0006] A halophilic Lactobacillus that catalyzes oxygen consumption in rocks during air drive in low-temperature oil reservoirs (Halolactibacillushalophilus ), is a halophilic lactobacillus ( Halolactibacillushalophilus YGgS-81, the halophilic lactobacillus ( Halolactibacillushalophilus YGgS-81 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 16, 2022, with accession number CGMCC No. 25748.

[0007] The halophilic lactobacillus ( Halolactibacillushalophilus The application of this technology in air-driven catalytic rock oxygen depletion in crude oil extraction, wherein the rock is a rock containing pyrite.

[0008] Preferably, in the air drive process, the *Halophilic Lactobacillus* (HAL) is injected into the reservoir simultaneously with the air injection. Halolactibacillushalophilus The fermentation broth was diluted and injected into the oil reservoir.

[0009] More preferably, the dilution method is as follows: The *Lactobacillus halophilus* (…) Halolactibacillushalophilus The fermentation broth was diluted with injection water to 15%~20wt% and then directly injected into the oil reservoir.

[0010] More preferably, the dilution method is as follows: The *Lactobacillus halophilus* (…) Halolactibacillushalophilus The fermentation broth is diluted to 5-10 wt% with injected water, then nutrients are added and mixed, and then injected into the oil reservoir to activate and multiply the halophilic lactobacillus in the oil reservoir; wherein, the nutrients corresponding to each liter of injected water are at least one of (NH4)2SO4 0.1-0.3g, K2HPO4·3H2O 2.8-3.2g, MgSO4·7H2O 0.4-0.6g, and CaCl2 0.18-0.20g.

[0011] Preferably, the fermentation broth is obtained by inoculating the seed culture of Lactobacillus halophilus onto a modified Waksman liquid medium and culturing it at 28-32°C and 160-180 rpm for 72-84 hours.

[0012] More preferably, the culture is carried out at 30°C and 160 rpm for 72 hours.

[0013] Preferably, the modified Waksman liquid culture medium is as follows: (NH4)2SO4 0.1-0.3g, MgSO4·7H2O 0.4-0.6g, K2HPO4·3H2O 2.8-3.2g, CaCl2·2H2O 0.18-0.20g, 1000mL deionized water, pH adjusted to 3.0-4.0 with 1:1 sulfuric acid solution, and autoclaved at 121℃ for 15 minutes; then 10g of elemental sulfur S 0After being sterilized by ultraviolet irradiation, it is mixed with the culture medium that has been sterilized by high pressure steam to obtain the modified Waksman liquid culture medium.

[0014] In this invention, halophilic lactobacillus ( Halolactibacillushalophilus Biological preservation information for YGgS-81: Date of deposit: September 16, 2022; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Address of depositary institution: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing; Accession number: CGMCC No. 25748, Institute of Microbiology, Chinese Academy of Sciences; Classification and nomenclature: Halophyte Lactobacillus ( Halolactibacillushalophilus ).

[0015] Advantages of this invention: The present invention provides halophilic lactobacillus ( Halolactibacillushalophilus YGgS-81 can utilize atmospheric CO2 as a carbon source to oxidize sulfur in pyrite within rocks and minerals. 2- Ions or elemental S 0 It obtains energy for growth and produces a large amount of acid during the growth process. This acid production accelerates the dissolution rate of pyrite in aqueous solution, allowing pyrite to decompose into Fe more quickly. 2+ and S 2- and further S 2- Oxidized to SO4 2- At the same time, H is continuously generated + Therefore, this bacterium can be used as a microbial catalyst in air drive or microbial-assisted air drive oil recovery, and has great application potential in ensuring the safety of air drive. Attached Figure Description

[0016] Figure 1 Phylogenetic tree of 16S rDNA of strain YGgS-81; Figure 2 The relationship between biomass and bioactivity of strain YGgS-81; Figure 3 The relationship between biomass and sulfur oxidation rate of strain YGgS-81; Figure 4 The sulfur oxidation rate of rock minerals in the test area by strain YGgS-81. Detailed Implementation

[0017] I. Culture medium formulation 1. Modified Waksman liquid medium: (NH4)2SO4 0.1-0.3g, MgSO47H2O 0.4-0.6g, K2HPO4·3H2O 2.8-3.2g, CaCl2·2H2O 0.18-0.20g, 1000mL deionized water, adjust pH to 3.0-4.0 with 1:1 sulfuric acid solution, autoclave at 121℃ for 15 minutes; then add 10g of elemental sulfur S 0 After being sterilized by ultraviolet irradiation, it is mixed with the culture medium that has been sterilized by high pressure steam to obtain the modified Waksman liquid culture medium.

[0018] 2. Starky-Na2S2O3 solid culture medium: Solution A: Add 2.0g of (NH4)2SO4, 3.0g of K2HPO4·3H2O, 0.5g of MgSO47H2O, and 0.25g of CaCl2·2H2O to 500mL of deionized water. Adjust the pH to 3.0 with a 1:1 sulfuric acid solution and autoclave at 121℃ for 15 minutes. Solution B: Add 0.001g of FeSO4·7H2O and 10g of Na2S2O3 to 300mL of deionized water and dissolve. Filter through a 0.22μm microporous membrane for sterilization. Solution C: Dissolve 15g of agar in 200mL of deionized water and autoclave at 121℃ for 15 minutes. After sterilization, wait for the temperature of solutions A and C to drop to 60℃ before mixing them with solution B.

[0019] 3. Does not contain S 0 or S 2- Enrichment medium: K2HPO4·3H2O 2.8~3.2g, MgSO4·7H2O 0.4-0.6g, CaCl2 0.18~0.20g, deionized water 1L, adjust the pH of the medium to 3-4 with 1:1 sulfuric acid solution, and autoclave at 121℃ for 15 minutes.

[0020] II. Halophilic Lactobacillus ( Halolactibacillushalophilus Screening of strains 1. Enrichment of bacterial strains Oil and water samples from the Baota block of the Yanchang Oilfield were centrifuged at 6000 rpm for 30 min within 24 hours of production. The precipitate was collected and inoculated into 100 mL of modified Waksman liquid medium. The samples were then incubated in a shaker at 30°C and 160 rpm until the pH of the medium in the conical flask dropped below 2.0. 10 mL of the culture was then transferred to 90 mL of fresh modified Waksman liquid medium for further incubation. This incubation process was repeated for 3-4 generations. 2. Isolation of single strains After acclimatizing the bacterial population for 3-4 generations, the resulting mixed bacterial population was diluted 0, 100, and 10000 times under aseptic conditions and spread onto Starky-Na2S2O3 solid medium (plates), which were then incubated upside down in a 30°C incubator. When round, milky-white or pale yellow colonies with a diameter of approximately 1 mm, neat edges, and a slightly convex center appeared on the solid medium, single colonies were picked and cultured in modified Waksman liquid medium at 30°C in a shaker. When the pH dropped below 2.0, the same 10-fold dilution method was repeated on the solid medium for streak isolation. After repeated purification, a single strain was isolated and named YGgS-81, which was then stored at 4°C. The modified Waksman liquid culture medium consisted of: (NH4)2SO4 0.2g, MgSO4·7H2O 0.5g, K2HPO4·3H2O 3.0g, CaCl2·2H2O 0.20g, 1000mL deionized water, pH adjusted to 3.0 with a 1:1 sulfuric acid solution, and autoclaved at 121℃ for 15 minutes; then 10g of elemental sulfur S... 0 After being sterilized by ultraviolet irradiation, it is mixed with the culture medium that has been sterilized by high pressure steam to obtain the modified Waksman liquid culture medium.

[0021] III. Identification of strain YGgS-81 The species of the isolated strain was determined by comparing its 16S rDNA sequence. The genome of strain YGgS-81 was extracted using the PowerSoil DNA Isolation Kit, and the 16S rDNA sequence was amplified by PCR using the following primers as a template: Upstream primer (F27): 5'-AGTTTGATCMTGGCTCAG-3'; Downstream primer (R1492): 5'-GGTTACCTTGT TACGACTT -3'; The PCR reaction system is as follows: 2×Pfu Master Mix 25μL Upstream primer 2μL, 2 μL of downstream primer 2μL template Add ddH2O to 50 μL; PCR reaction conditions: pre-denaturation 94℃ 3 min; 30 cycles: denaturation 94℃ 30 s, annealing 57℃ 30 s, extension 72℃ 2 min; subsequent extension 72℃ 5 min, stop reaction at 4℃ and incubate. The obtained PCR products were recovered and purified, then sent to Shanghai Meiji Biotechnology Co., Ltd. for Sanger sequencing. The resulting sequences were BLASTed (NCBI) and a phylogenetic tree was constructed using MEGA software, as shown below. Figure 1 As shown, strain YGgS-81 was ultimately identified as belonging to the genus *Haloxylactobacillus*. Halolactibacillushalophilus ), named *Lactobacillus halophilus* ( Halolactibacillushalophilus )YGgS-81.

[0022] The strain YGgS-81 was deposited on September 16, 2022, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCCNo. 25748, and classified as *Lactobacillus halophilus*. Halolactibacillushalophilus ).

[0023] IV. Sulfur oxidation activity of strain YGgS-81 (1) Seed culture: After activating the strain YGgS-81 stored at 4℃, it was inoculated into 100mL of modified Waksman liquid medium and cultured overnight at 30℃ and 160rpm until the logarithmic growth phase to obtain the seed culture of Lactobacillus halophilus YGgS-81. (2) Determination of sulfur oxidation activity: All seed culture was filtered through neutral qualitative filter paper. The filtrate was centrifuged at 10000×g, and the lower part of the cells was collected. The cells were washed three times with sulfuric acid aqueous solution at pH=3 to remove impurity ions. After centrifugation again, the precipitate was resuspended in 100mL of sulfuric acid aqueous solution. The inoculum was added to the sulfuric acid aqueous solution at pH=3 at inoculum rates of 0%, 5%, 10%, 15%, and 20% (v / v). Then, elemental sulfur was added at 10g / L, and the mixture was cultured at 30℃ and 160rpm for 7 days with constant temperature shaking. After the culture was completed, the reaction solution was centrifuged at 8000rpm / min for 5 minutes, and the supernatant was collected. The pH of the reaction solution was measured using a pH meter. Figure 2 Simultaneously, ion chromatography was used to determine the SO4 content in the reaction solution. 2- Concentration, calculate the oxidation rate of sulfur in the reaction system ( Figure 3 ); without adding Lactobacillus halophilus ( Halolactibacillushalophilus The control group consisted of samples with an inoculation rate of 0%. The final pH values ​​of the reaction solution were investigated when the bacterial biomass was 5%, 10%, 15%, and 20% (v / v). Figure 2 ) and the oxidation rate of sulfur in the reaction solution ( Figure 3 It was found that as the biomass of *Lactobacillus halosa* increased, the pH of the reaction solution gradually decreased. At a biomass of 15%, the pH of the reaction solution dropped from an initial 3.0 to approximately 1.2. This indicates that *Lactobacillus halosa* produces a large amount of H₂ while continuously multiplying. +This lowers the pH of the reaction solution. Simultaneously, the concentration of SO4 in the reaction solution gradually increases, and the greater the biomass, the higher the concentration. 2- The faster the concentration increases, the larger the biomass of *Lactobacillus halophilus*, the stronger its catalytic activity in oxidizing elemental sulfur, and the higher the oxidation rate of elemental sulfur. When the biomass of *Lactobacillus halophilus* is 15%, the oxidation rate of elemental sulfur after the reaction can reach 91.2%, which is 84% ​​higher than the 7.2% of the control group.

[0024] This indicates that the biomass of *Lactobacillus halophilus* is the main controlling factor for the oxidation rate and efficiency of sulfur in rocks and minerals. When the biomass is 15-20%, the oxidation rate of sulfur is relatively high.

[0025] V. Tests on the bio-oxidation of rocks and minerals in the experimental area by strain YGgS-81 Will not contain S 0 or S 2- Rock samples (rich in pyrite) from the Yanchang Oilfield Baota Block test area were mixed with 4 g / L of 85% (v / v) and 15% (v / v) halophilic Lactobacillus YGgS-81 seed culture medium (particle size 1-2 mm). The mixture was then incubated at 30℃ with shaking at 160 r / min for at least 30 days. During the incubation period, 5 mL of the reaction solution was collected on days 0, 5, 10, 15, 20, 25, and 30, and centrifuged at 8000 rpm for 5 minutes. The supernatant was collected, and SO42- in the reaction system was determined by ion chromatography. 2- The concentration was determined by using the solution without *Lactobacillus halophilus* YGgS-81 seed culture as a control. (See...) Figure 4 ; wherein, the one that does not contain S 0 or S 2- The enrichment medium is as follows: (NH4)2SO4 0.2g, K2HPO4·3H2O 3.0g, MgSO4·7H2O 0.5g, CaCl2 0.20g, deionized water 1L, the medium pH is adjusted to 3 with 1:1 sulfuric acid solution, and sterilized at 121℃ for 15 minutes by high temperature and high pressure.

[0026] The results showed that with prolonged reaction time, 30-day testing revealed that *Lactobacillus halophilus* significantly increased the oxidation rate in rock minerals, compared to the untreated control, SO42-... 2- The concentration was 5 times that of the control group without added Lactobacillus halophilus. Figure 4 Therefore, it can be concluded that halophilic lactobacillus can accelerate the oxidation rate of sulfur in rocks and minerals.

[0027] Lactobacillus salina ( HalolactibacillushalophilusYGgS-81 is a chemoautotrophic microorganism that utilizes atmospheric CO2 as a carbon source. It obtains the energy needed for growth by oxidizing ions in pyrite-rich rocks and minerals, accelerating the rapid oxidation of pyrite and consuming atmospheric oxygen during its growth. Therefore, in air-driven enhanced oil recovery, the bio-oxidation of rocks and minerals by this bacterium can be utilized to rapidly consume atmospheric oxygen, ensuring safe production in air-driven reservoirs. During air-driven enhanced oil recovery, the *Lactobacillus halophilus* (a type of microorganism) is injected into the reservoir simultaneously with air injection. Halolactibacillushalophilus The fermentation broth is diluted and injected into the oil reservoir; the dilution method is any one of the following two methods: a. The halophilic lactobacillus ( Halolactibacillushalophilus The fermentation broth was diluted with injection water to 15%~20wt% and then directly injected into the oil reservoir; b. The halophilic lactobacillus ( Halolactibacillushalophilus The fermentation broth is diluted to 5-10 wt% with injected water, then nutrients are added and mixed, and then injected into the oil reservoir to activate and multiply the halophilic lactobacillus in the oil reservoir; wherein, the nutrients corresponding to each liter of injected water are at least one of (NH4)2SO4 0.1-0.3g, K2HPO4·3H2O 2.8-3.2g, MgSO4·7H2O 0.4-0.6g, and CaCl2 0.18-0.20g.

[0028] Preferably, the fermentation broth is as follows: seed culture of Lactobacillus halophilus is inoculated onto fresh modified Waksman liquid medium at an inoculation rate of 5-20%, and cultured at 28-32℃ and 160-180 rpm for 72-84 hours to obtain the fermentation broth.

[0029] More preferably, the fermentation broth is as follows: seed culture of Lactobacillus halophilus is inoculated onto fresh modified Waksman liquid medium at an inoculation rate of 15-20%, and cultured at 30°C and 160 rpm for 72 h to obtain the fermentation broth.

Claims

1. A halophilic Lactobacillus species that catalyzes oxygen consumption in low-temperature oil reservoirs via air drive ( Halolactibacillus halophilus ), characterized in that: It is a halophilic lactobacillus ( Halolactibacillus halophilus YGgS-81, the halophilic lactobacillus ( Halolactibacillus halophilus YGgS-81 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 16, 2022, with accession number CGMCC No. 25748.

2. The *Halophilic Lactobacillus* as described in claim 1 ( Halolactibacillus halophilus Its application in air-driven catalytic rock oxygen depletion in crude oil extraction is characterized by: The rock in question is a rock containing pyrite.

3. The *Lactobacillus halophilus* according to claim 2 ( Halolactibacillus halophilus Its application in air-driven catalytic rock oxygen depletion in crude oil extraction is characterized by: During air drive, air is injected into the reservoir while simultaneously introducing the halophilic lactobacillus (…). Halolactibacillus halophilus The fermentation broth was diluted and injected into the oil reservoir.

4. The halophilic lactobacillus according to claim 3 ( Halolactibacillus halophilus Its application in air-driven catalytic rock oxygen depletion in crude oil extraction is characterized by: The halophilic lactobacillus ( Halolactibacillus halophilus The fermentation broth was diluted with injection water to 15%~20wt% and then directly injected into the oil reservoir.

5. The halophilic lactobacillus according to claim 3 ( Halolactibacillus halophilus Its application in air-driven catalytic rock oxygen depletion in crude oil extraction is characterized by: The halophilic lactobacillus ( Halolactibacillus halophilus The fermentation broth is diluted to 5-10 wt% with injection water, then nutrients are added and mixed before being injected into the oil reservoir. The nutrients per liter of injection water are at least one of the following: (NH4)2SO4 0.1-0.3 g, K2HPO4·3H2O 2.8-3.2 g, MgSO4·7H2O 0.4-0.6 g, and CaCl2 0.18-0.20 g.

6. The *Halophilic Lactobacillus* according to claim 3, 4, or 5 ( Halolactibacillus halophilus Its application in air-driven catalytic rock oxygen depletion in crude oil extraction is characterized by: The fermentation broth is obtained by inoculating the seed culture of Lactobacillus halophilus onto a modified Waksman liquid medium and culturing it at 28-32℃ and 160-180rpm for 72-84h.

7. The *Halophilic Lactobacillus* according to claim 6 ( Halolactibacillus halophilus Its application in air-driven catalytic rock oxygen depletion in crude oil extraction is characterized by: Incubate at 30℃ and 160 rpm for 72 h.

8. The halophilic lactobacillus according to claim 7 ( Halolactibacillus halophilus Its application in air-driven catalytic rock oxygen depletion in crude oil extraction is characterized by: The modified Waksman liquid medium is as follows: (NH4)2SO4 0.1-0.3g, MgSO4·7H2O 0.4-0.6g, K2HPO4·3H2O 2.8-3.2g, CaCl2·2H2O 0.18-0.20g, 1000mL deionized water, pH adjusted to 3.0-4.0 with 1:1 sulfuric acid solution, and autoclaved at 121℃ for 15 minutes; then 10g of elemental sulfur (S) sterilized by ultraviolet irradiation is mixed with the autoclaved medium to obtain the modified Waksman liquid medium.

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