Denitrifying bacteria and application thereof in biological desulfurization in oil field system

Abstract

The invention relates to denitrifying bacteria and application thereof in biological desulfurization in an oil field system. The denitrifying bacteria are named as warm bacillus V3, and the preservation number of the denitrifying bacteria is CGMCC (China General Microbiological Culture Collection Center) NO.46386. The warm bacillus V3 and sodium nitrate are injected into an oil field system containing sulfate reducing bacteria in a water injection mode for biological sulfur removal. Compared with the prior art, a novel denitrifying bacterium capable of remarkably reducing the sulfur ions in the water body of the oil field system is innovatively found, the novel denitrifying bacterium belongs to a new species of thermobacillus, and the highest removal rate of the sulfur ions can reach 99.2% after the denitrifying bacterium is added into an oil field sulfate reducing bacterium system for short-term culture.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a denitrifying bacterium and its application in biological desulfurization in an oilfield system. Background Art

[0002] In related fields such as oil and gas field exploitation, the problem of hydrogen sulfide corrosion caused by microorganisms is widespread. During the water injection development process in many oilfields, since the reinjected water contains certain nutrients such as sulfates, it provides conditions for the growth of microorganisms such as sulfate-reducing bacteria, thereby generating hydrogen sulfide and causing corrosion, which greatly increases the maintenance cost and safety risk. Therefore, the negative impact brought by sulfate-reducing bacteria is one of the problems that must be addressed and solved during the oil and gas field development process.

[0003] Due to the many negative impacts brought by the corrosion caused by sulfate-reducing bacteria, multiple strategies are needed to alleviate the corrosion, including chemical methods, physical methods, and biological methods. Among them, the biological method is widely used due to its green safety, good inhibition efficiency, etc. One method is to inject nitrate into the reservoir to stimulate the growth of denitrifying bacteria, and the nitrite produced by the denitrifying bacteria inhibits the growth of sulfate-reducing bacteria, significantly reducing the hydrogen sulfide concentration and the pipeline corrosion rate.

[0004] Although there are currently control methods for the corrosion caused by hydrogen sulfide produced by microorganisms in the reservoir environment, they still face challenges such as a long inhibition period and a low inhibition rate. For example, the prior art CN118185779A provides a method for preventing and controlling sulfate-reducing bacteria in oil and gas gathering pipelines using an anaerobic denitrifying bacterium and its bacterial agent. This method uses a Klebsiella oxytoca to inhibit sulfate-reducing bacteria, with an inhibition period of 30 days and a maximum hydrogen sulfide removal rate of 97.9%; the prior art CN104357035A provides a biological bacterial agent for preventing and controlling SRB in high-temperature water bodies and a method for inhibiting SRB. This method uses a Geobacillus to inhibit sulfate-reducing bacteria, with an inhibition period of 60 days and a maximum hydrogen sulfide removal rate of 97.73%. The above prior art has a long inhibition period and is difficult to meet the rapid inhibition demand for the activity of sulfate-reducing bacteria in the oilfield site.

[0005] In addition, biological inhibitors are also a widely used anti-corrosion strategy. The prior art CN102090420A provides a method for biologically inhibiting the generation of secondary hydrogen sulfide by sulfate-reducing bacteria in a crude oil gathering system. The biological inhibitors used in this method are sodium nitrite, humic acid, sodium borate, sodium hydroxide, and glutaraldehyde, with a maximum hydrogen sulfide removal rate of 95.8%. Although the biological inhibitors used in the above prior art can achieve the anti-corrosion effect, problems such as complex inhibitors, high costs, and poor inhibition effects still exist.

[0006] Therefore, there is an urgent need for a sulfate-reducing bacteria inhibitor with a short inhibition period and a high hydrogen sulfide removal rate, so as to effectively control the corrosion problem caused by hydrogen sulfide produced by microorganisms in the reservoir environment. Summary of the Invention

[0007] The purpose of the present invention is to provide a denitrifying bacterium and its application in biological desulfurization in the oilfield system to overcome the defects of the existing technologies described above.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] One of the technical solutions of the present invention is to provide a denitrifying bacterium named Bacillus thermosphaericus V3, and its preservation number is CGMCC NO. 46386.

[0010] Another technical solution of the present invention is to provide a bacterial agent, which includes Bacillus thermosphaericus V3 described in the above technical solution, and its preservation number is CGMCC NO. 46386.

[0011] In some specific embodiments, the bacterial agent is a bacterial suspension or dry bacterial cells.

[0012] Another technical solution of the present invention is to provide a preparation method of the bacterial agent described in the above technical solution, including the following steps:

[0013] Inoculate Bacillus thermosphaericus V3 with the preservation number of CGMCC NO. 46386 into a denitrification medium, and carry out anaerobic fermentation at 30-50 °C to prepare a fermentation broth; obtain dry bacterial cells through centrifugation and freeze-drying, or obtain a bacterial suspension through centrifugation and diluting the bacteria.

[0014] In some specific embodiments, the denitrification medium includes:

[0015] NaCl 2.85 g / L, MgCl2·6H2O 0.28 g / L, CaCl2 0.55 g / L, NH4Cl 0.24 g / L, KCl 0.10 g / L, yeast extract 0.5 g / L, NaNO3 0.5 g / L, Na2S·9H2O 0.4 g / L, NaHCO3 0.2 g / L, KH2PO4 0.2 g / L and trace elements 5.0 mL / L,

[0016] The trace elements include:

[0017] 1.5 g / L of nitrilotriacetic acid, 3.0 g / L of MgSO4·7H2O, 0.5 g / L of MnSO4·H2O, 1.0 g / L of NaCl, 0.1 g / L of FeSO4·7H2O, 0.18 g / L of CoSO4·7H2O, 0.1 g / L of CaCl2·2H2O, 0.18 g / L of ZnSO4·7H2O, 0.01 g / L of CuSO4·5H2O, 0.02 g / L of KAl(SO4)2·12H2O, 0.01 g / L of H3BO3, 0.01 g / L of NaMoO4·2H2O, 0.03 g / L of NiCl2·6H2O.

[0018] In a specific embodiment, the anaerobic culture temperature is 37 °C.

[0019] The fourth technical solution of the present invention is to provide an application of the denitrifying bacteria as described in one of the above technical solutions in preventing and controlling sulfate-reducing bacteria.

[0020] In some specific embodiments, the denitrifying bacteria are used to prevent and control sulfate-reducing bacteria in the oilfield system.

[0021] The fifth technical solution of the present invention is to provide a method for preventing and controlling sulfate-reducing bacteria in the oilfield system by using the denitrifying bacteria as described in one of the above technical solutions, which includes the following steps:

[0022] Inject Bacillus thermoglucosidasius V3 and sodium nitrate into the oilfield system containing sulfate-reducing bacteria by water injection for biological desulfurization.

[0023] In some specific embodiments, the inoculation amount of Bacillus thermoglucosidasius V3 is 5% - 10% of the volume of the oilfield system containing sulfate-reducing bacteria, and the addition amount of sodium nitrate is 0.30 - 0.50 g / L of the oilfield system containing sulfate-reducing bacteria.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention innovatively discovers a novel denitrifying bacterium that can significantly reduce the sulfur ions in the water body of the oilfield system. It belongs to a new species of the genus Bacillus thermoglucosidasius and is named Bacillus thermoglucosidasius V3, with the preservation number of CGMCC NO.46386, providing a new strain selection for the anti-corrosion field.

[0026] (2) The present invention provides a method for biological desulfurization in the oilfield system, which shortens the culture cycle and effectively reduces the content of sulfides in the water body of the oilfield system. After Bacillus thermoglucosidasius V3 provided by the present invention is added to the oilfield sulfate-reducing bacteria system for short-term culture, the removal rate of sulfur ions can reach up to 99.2%.

[0027] (3) The method for biological desulfurization in an oilfield system provided by the present invention has a simple system operation, is green and safe, and has outstanding application effects, showing broad application prospects in the field of corrosion prevention and control in the oilfield system. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It shows the removal of sulfide ions in the Tepidibacillus sp. V3 system.

[0029] Figure 2 It shows the inhibition of Desulfovibrio desulfuricans by the Tepidibacillus sp. V3 system.

[0030] Figure 3 It shows the inhibition of sulfate-reducing bacteria by the Tepidibacillus sp. V3 system. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] In the present invention, Tepidibacillus sp. V3 has a deposit number of CGMCC NO. 46386 and was deposited on March 10, 2025, at the General Microbiology Center of the China Microbial Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] The present invention will be described in detail below with reference to the drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0033] In the following embodiments, unless otherwise specified for raw materials or treatment techniques, it means that they are all conventional commercially available raw material products or conventional treatment techniques in the art.

[0034] Example 1

[0035] This example is for the isolation, screening and verification of Tepidibacillus sp. V3

[0036] Using the oilfield produced water sample as the separation source, the Hungate anaerobic roll tube separation method was used for single bacterium separation. The anaerobic roll tubes were placed in an oven at 37°C for dark incubation, and then monoclonal colonies were picked and subjected to strain identification. The medium used for separation was: NaCl 2.85 g / L, MgCl2·6H2O 0.28 g / L, CaCl2 0.55 g / L, NH4Cl 0.24 g / L, KCl 0.10 g / L, yeast extract 0.5 g / L, NaNO3 0.5 g / L, Na2S·9H2O 0.4 g / L, NaHCO3 0.2 g / L, KH2PO4 0.2 g / L, and trace elements 5.0 mL / L; the formula for trace elements was: nitrilotriacetic acid 1.5 g / L, MgSO4·7H2O 3.0 g / L, MnSO4·H2O 0.5 g / L, NaCl 1.0 g / L, FeSO4·7H2O 0.1 g / L, CoSO4·7H2O 0.18 g / L, CaCl2·2H2O 0.1 g / L, ZnSO4·7H2O 0.18 g / L, CuSO4·5H2O 0.01 g / L, KAl(SO4)2·12H2O 0.02 g / L, H3BO3 0.01 g / L, NaMoO4·2H2O 0.01 g / L, NiCl2·6H2O 0.03 g / L.

[0037] Molecular biological identification was carried out on the isolated single bacterium. The specific classification identification primers 27F and 1492R for bacteria were used to amplify the fragments of 16S rDNA of the strain respectively, and the amplified sequences were sequenced and compared. The sequences of 27F and 1492R are shown in SEQ ID NO.1 and 2 respectively:

[0038] 27F (SEQ ID NO.1): 5’-AGAGTTTGATCCTGGCTCAG-3’;

[0039] 1492R (SEQ ID NO.2): 5’-GGTTACCTTGTTACGACTT-3’

[0040] After sequencing and comparing the amplified sequences, it was found that the similarity with the sequence of Bacillus thermophilus was the highest, and the similarity range was between 94% and 95%. Based on the phylogenetic tree, it was determined that this bacterium belongs to a new species of the genus Bacillus thermophilus in taxonomy, and it was named Bacillus thermophilus V3.

[0041] Example 2

[0042] This example was for the growth and metabolism verification of Bacillus thermophilus V3

[0043] Bacillus thermoglucosidasius V3 was inoculated into a medium and anaerobically cultured at 37°C. The composition of the medium was as follows: 2.85 g / L of NaCl, 0.28 g / L of MgCl2·6H2O, 0.55 g / L of CaCl2, 0.24 g / L of NH4Cl, 0.10 g / L of KCl, 0.5 g / L of yeast extract, 0.5 g / L of NaNO3, 0.4 g / L of Na2S·9H2O, 0.2 g / L of NaHCO3, 0.2 g / L of KH2PO4, and 5.0 mL / L of trace elements; the formula of the trace elements was as follows: 1.5 g / L of nitrilotriacetic acid, 3.0 g / L of MgSO4·7H2O, 0.5 g / L of MnSO4·H2O, 1.0 g / L of NaCl, 0.1 g / L of FeSO4·7H2O, 0.18 g / L of CoSO4·7H2O, 0.1 g / L of CaCl2·2H2O, 0.18 g / L of ZnSO4·7H2O, 0.01 g / L of CuSO4·5H2O, 0.02 g / L of KAl(SO4)2·12H2O, 0.01 g / L of H3BO3, 0.01 g / L of NaMoO4·2H2O, 0.03 g / L of NiCl2·6H2O.

[0044] Figure 1 This shows the situation of sulfur ion removal by Bacillus thermoglucosidasius V3. After 14 days of culture, the sulfur ion concentration in the medium decreased significantly and was almost completely consumed, and the sulfur ion removal rate was 91.5%. This indicates that the isolated Bacillus thermoglucosidasius V3 can oxidize sulfides for growth and metabolism.

[0045] Example 3

[0046] This example is about the application of the Bacillus thermoglucosidasius V3 inhibition system.

[0047] Bacillus thermophilus V3 was inoculated into a medium and anaerobically cultured at 37 °C for 7 days. The medium composition was: NaCl 2.85 g / L, MgCl2·6H2O 0.28 g / L, CaCl2 0.55 g / L, NH4Cl 0.24 g / L, KCl 0.10 g / L, yeast extract 0.5 g / L, NaNO3 0.5 g / L, Na2S·9H2O 0.4 g / L, NaHCO3 0.2 g / L, KH2PO4 0.2 g / L, and trace elements 5.0 mL / L; the trace element formulation was: nitrilotriacetic acid 1.5 g / L, MgSO4·7H2O 3.0 g / L, MnSO4·H2O 0.5 g / L, NaCl 1.0 g / L, FeSO4·7H2O 0.1 g / L, CoSO4·7H2O 0.18 g / L, CaCl2·2H2O 0.1 g / L, ZnSO4·7H2O 0.18 g / L, CuSO4·5H2O 0.01 g / L, KAl(SO4)2·12H2O 0.02 g / L, H3BO3 0.01 g / L, NaMoO4·2H2O 0.01 g / L, NiCl2·6H2O 0.03 g / L.

[0048] The bacterial solution of Desulfovibrio (a type of sulfate-reducing bacteria (SRB)) from the produced water of Jiangsu Oilfield was inoculated into a basal medium at a volume ratio of 5%. The basal medium composition was: NaCl 2.85 g / L, MgCl2·6H2O 0.28 g / L, CaCl2 0.55 g / L, NH4Cl 0.24 g / L, KCl 0.10 g / L, yeast extract 0.5 g / L, NaNO3 0.5 g / L, Na2S·9H2O 0.4 g / L, NaHCO3 0.2 g / L, KH2PO4 0.2 g / L, and trace elements 5.0 mL / L; the trace element formulation was: nitrilotriacetic acid 1.5 g / L, MgSO4·7H2O 3.0 g / L, MnSO4·H2O 0.5 g / L, NaCl 1.0 g / L, FeSO4·7H2O 0.1 g / L, CoSO4·7H2O 0.18 g / L, CaCl2·2H2O 0.1 g / L, ZnSO4·7H2O 0.18 g / L, CuSO4·5H2O 0.01 g / L, KAl(SO4)2·12H2O 0.02 g / L, H3BO3 0.01 g / L, NaMoO4·2H2O 0.01 g / L, NiCl2·6H2O 0.03 g / L. Then sodium nitrate (added at a final concentration of 0.30 g / L) and the bacterial solution of Bacillus thermophilus V3 with an inoculation amount of 5% were added to the Desulfovibrio bacterial solution. At the same time, a control group without adding any inhibition system, that is, a control group only inoculated with Desulfovibrio, was set up, and 3 parallel samples were set for each group. AsFigure 2 As shown in the figure, after anaerobic culture at 37°C for 12 days, it was found that the removal rate of sulfide ions in the water body was approximately 99.2%.

[0049] Example 4

[0050] This example is about the application of the Bacillus stearothermophilus V3 inhibition system.

[0051] Bacillus stearothermophilus V3 was inoculated into the culture medium and anaerobically cultured at 37°C for 7 days. The composition of the culture medium was: NaCl 2.85 g / L, MgCl2·6H2O 0.28 g / L, CaCl2 0.55 g / L, NH4Cl 0.24 g / L, KCl 0.10 g / L, yeast extract 0.5 g / L, NaNO3 0.5 g / L, Na2S·9H2O 0.4 g / L, NaHCO3 0.2 g / L, KH2PO4 0.2 g / L, and trace elements 5.0 mL / L; the formula of the trace elements was: nitrilotriacetic acid 1.5 g / L, MgSO4·7H2O 3.0 g / L, MnSO4·H2O 0.5 g / L, NaCl 1.0 g / L, FeSO4·7H2O 0.1 g / L, CoSO4·7H2O 0.18 g / L, CaCl2·2H2O 0.1 g / L, ZnSO4·7H2O 0.18 g / L, CuSO4·5H2O 0.01 g / L, KAl(SO4)2·12H2O 0.02 g / L, H3BO3 0.01 g / L, NaMoO4·2H2O 0.01 g / L, NiCl2·6H2O 0.03 g / L.

[0052] The sulfate-reducing bacteria enriched bacterial liquid enriched from the produced water of Jiangsu Oilfield was inoculated into the basal medium at a volume ratio of 5%. The composition of the basal medium was: NaCl 2.85 g / L, MgCl₂·6H₂O 0.28 g / L, CaCl₂ 0.55 g / L, NH₄Cl 0.24 g / L, KCl 0.10 g / L, yeast extract 0.5 g / L, NaNO₃ 0.5 g / L, Na₂S·9H₂O 0.4 g / L, NaHCO₃ 0.2 g / L, KH₂PO₄ 0.2 g / L and trace elements 5.0 mL / L; the formula of the trace elements was: nitrilotriacetic acid 1.5 g / L, MgSO₄·7H₂O 3.0 g / L, MnSO₄·H₂O 0.5 g / L, NaCl 1.0 g / L, FeSO₄·7H₂O 0.1 g / L, CoSO₄·7H₂O 0.18 g / L, CaCl₂·2H₂O 0.1 g / L, ZnSO₄·7H₂O 0.18 g / L, CuSO₄·5H₂O 0.01 g / L, KAl(SO₄)₂·12H₂O 0.02 g / L, H₃BO₃ 0.01 g / L, NaMoO₄·2H₂O 0.01 g / L, NiCl₂·6H₂O 0.03 g / L. Then sodium nitrate (added at a final concentration of 0.50 g / L) and a warm Bacillus sp. V3 bacterial liquid with an inoculation amount of 10% were added to the sulfate-reducing enriched bacterial liquid. At the same time, a control group without adding any inhibition system was set up, that is, a control group only inoculated with the sulfate-reducing bacteria enriched bacterial liquid, and 3 parallel samples were set up for each group. As Figure 3 shown, after anaerobic culture at 37 °C for 12 days, it was found that the removal rate of sulfide ions in the water body was about 96.2%.

[0053] In the experimental verification stage of the inhibition system of Bacillus sp. V3 constructed in the present invention, by reproducing the key elements of the life activities of sulfate-reducing bacteria in the oilfield system (including temperature, electron donors and electron acceptors), the sulfate-reducing bacteria in the water body were enriched. After adding the Bacillus sp. V3 system, the sulfides in the oilfield system were effectively removed. Therefore, the inhibition system of Bacillus sp. V3 constructed in this experiment realized the immediate application of preventing and controlling the microbial corrosion risk in the oilfield system, significantly reduced the pipeline corrosion problems and potential safety hazards caused by sulfides such as hydrogen sulfide, and ensured the stable operation of the oilfield production system.

[0054] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A denitrifying bacterium, characterized in that: It was named as Bacillus thermophilus V3, and its deposit number is CGMCCNO.46386.

2. A bacterial agent, characterized in that It comprises the thermophilic Bacillus V3 as claimed in claim 1, and its deposit number is CGMCC NO.46386.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is a bacterial suspension or bacterial dry powder.

4. A method for preparing the bacterial agent as claimed in claim 2 or 3, characterized in that: The steps include: The thermophilic bacillus V3 with a preservation number of CGMCC NO.46386 is inoculated into a denitrification medium, and anaerobically fermented at 30-50°C to prepare a fermentation liquid; a bacterial dry powder is obtained by centrifugation and freeze-drying, or a bacterial suspension is obtained by centrifugation and dilution of the bacterial cells.

5. The preparation method according to claim 4, characterized in that: The denitrification culture medium comprises: NaCl 2.85g / L, MgCl2·6H2O 0.28g / L, CaCl2 0.55g / L, NH4Cl 0.24g / L, KCl 0.10g / L, yeast extract 0.5g / L, NaNO3 0.5g / L, Na2S·9H2O 0.4g / L, NaHCO3 0.2g / L, KH2PO4 0.2g / L and trace elements 5.0mL / L, The trace elements include: Nitrilotriacetic acid 1.5g / L, MgSO4·7H2O 3.0g / L, MnSO4·H2O 0.5g / L, NaCl 1.0g / L, FeSO4·7H2O 0.1g / L, CoSO4·7H2O 0.18g / L, CaCl2·2H2O 0.1g / L, ZnSO4·7H2O 0.18g / L, CuSO4·5H2O 0.01g / L, KAl(SO4)2·12H2O 0.02g / L, H3BO3 0.01g / L, NaMoO4·2H2O 0.01g / L, NiCl2·6H2O 0.03g / L.

6. The preparation method according to claim 4, characterized in that: The anaerobic culture temperature was 37°C.

7. Use of the denitrifying bacteria as claimed in claim 1 in controlling sulfate-reducing bacteria.

8. The use according to claim 7, characterized in that: The denitrifying bacteria are used to control sulfate-reducing bacteria in oil field systems.

9. The method for controlling sulfate-reducing bacteria in an oil field system using denitrifying bacteria according to claim 1, characterized in that: The steps include: Thermobacillus sp. V3 and sodium nitrate were injected into the oil field system containing sulfate-reducing bacteria through water injection for biological desulfurization.

10. The method according to claim 9, characterized in that The inoculation amount of the thermobacillus V3 is 5% to 10% of the volume of the oil field system containing sulfate-reducing bacteria, and the addition amount of the sodium nitrate is 0.30 to 0.50 g / L of the oil field system containing sulfate-reducing bacteria.

Images