Citrobacter freundii for biotransformation of carbon dioxide in oil reservoirs and application thereof

Abstract

This invention discloses a Citrobacter freundii strain for methanogenesis from carbon dioxide bioconversion in oil reservoirs. Citrobacter freundii The *Citrobacter freundii*, deposited on September 16, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 25749, was also provided in this invention. Citrobacter freundii Application of Citrobacter freundii in oil reservoir CO2 bioconversion to methanogenesis. This invention provides Citrobacter freundii (… Citrobacter freundii This strain is a microorganism that can improve the bioconversion of CO2 to methane. It can still grow actively in the reservoir environment and convert CO2 into CH4. It has great application potential in the fields of reservoir CO2 storage and bioconversion to CH4.

Description

Technical Field

[0001] This invention belongs to the field of oilfield microbial technology, specifically relating to a Citrobacter freundii strain that produces methanates from carbon dioxide bioconversion in oil reservoirs and its application in improving CO2 bioconversion to methanates in oil reservoirs. Background Technology

[0002] Carbon capture, utilization, and storage (CCUS) is a key measure aimed at achieving significant emissions reductions from fossil fuels, and it is projected to contribute one-third of global CO2 emission reductions by 2050. Currently, large-scale CO2 sequestration methods mainly include geological sequestration and mineralized sequestration. Suitable sites for CO2 geological sequestration include deep saline aquifers, depleted oil and gas reservoirs, coal seams, and oil and gas reservoirs currently in production. Oil reservoirs act as natural geological anaerobic bioreactors, where various microorganisms can synergistically convert injected and sequestered CO2 into CH4 through biological pathways. CH4 is currently the cleanest hydrocarbon energy source in the environment and is the most promising potential conversion product.

[0003] Oil reservoirs are natural sites for large-scale CO2 geological sequestration. After CO2 sequestration, the carbon element in the CO2 in the reservoir remains unstable for thousands of years before it is completely chemically trapped. Therefore, it can be converted into high-value-added products such as CH4 through microbial technology. This technology can greatly enhance the exploitation value and life cycle of depleted oil and gas reservoirs.

[0004] Previous studies have demonstrated the feasibility and significant application potential of CO2 fixation and bioconversion to CH4 technology in oil reservoirs. Current experiments have shown that injecting inorganic nutrients into oil reservoirs can activate methanogenic bacteria, thereby converting CO2 into CH4 and realizing the resource utilization of CO2. This technology not only converts residual CO2 in oil reservoirs, increasing their economic value, but also extends the exploitation lifecycle of depleted oil and gas reservoirs. Methanogenic bacteria play a crucial role in the natural carbon cycle, encompassing more than 10 families and over 30 genera, including Methanobacteria, Methanococci, Methanomicrobes, Methanosporidiales, Methanogenic Volcaniculales, and Methanocytozoa.

[0005] The future research and development of CO2 bioconversion CH4 technology in oil reservoir environments will focus on the following aspects: (1) screening, optimizing and activating relevant advantageous strains and inhibiting the competitive bacterial community of methanogens; (2) clarifying the main strains and interrelationships between bacterial communities designed for this technology in order to construct and control a reasonable bacterial community structure; (3) clarifying the main limiting factors of methanogen methanogenesis and its rate-limiting reaction in order to improve the reaction rate and CO2 conversion rate. Summary of the Invention

[0006] This invention provides a Citrobacter freundii strain for bioconversion of carbon dioxide to methanogens in oil reservoirs and its application in improving bioconversion of CO2 to methanogens in oil reservoirs.

[0007] A type of Citrobacter freundii that bioconverts carbon dioxide to produce methanogens in oil reservoirs ( Citrobacter freundii (This refers to Citrobacter freundii) (Citrobacter freundii Strain YGgY-138 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 16, 2022, with accession number CGMCC No. 25749.

[0008] The Citrobacter freundii ( Citrobacter freundii In the application of CO2 bioconversion to produce methane in oil reservoirs, the reservoirs can be used for CO2 flooding or sequestration.

[0009] Preferably, the application is carried out during CO2 flooding or sequestration, where the *Citrobacter freundii* (Citrobacter freundii) is injected into the reservoir simultaneously with CO2. Citrobacter freundii The seed solution was diluted with water to 5-10 wt% and then injected into the oil reservoir.

[0010] Preferably, after dilution, nutrients are added before injection into the reservoir; wherein each liter of injected water contains the following nutrients: 0.3-0.5g K2HPO4·3H2O, 0.1-0.3g KH2PO4, 0.8-1.2g NH4Cl, 0.08-0.12g MgCl2·6H2O, 1.5-2.5g sodium acetate, 1.5-2.5g sodium formate, 0.8-1.2g yeast extract, 0.8-1.2g tryptone, 0.4-0.6g L-cysteine ​​hydrochloride, 0.8-1.2mL 0.1% resazurite, 0.8-1.2mL trace element concentrate, 0.8-1.2mL compound vitamin concentrate, 18-22mL 1% Na2S·9H2O, and 8-12mL 10% NaHCO3.

[0011] Preferably, the trace element concentrate is as follows: 10.0-14.0 g of aminotriacetic acid, 1.2-1.5 g of FeCl3·6H2O, 0.020-0.026 g of CoCl2·6H2O, 0.08-0.12 g of CaCl2·2H2O, 0.08-0.12 g of MnCl2·4H2O, 0.08-0.12 g of ZnCl2, 0.020-0.030 g of CuCl2·2H2O, 0.08-0.12 g of H3BO3, 0.022-0.026 g of Na2MoO4·2H2O, 0.8-1.2 g of NaCl, and NiCl2·6H2O. Dissolve 0.10-0.14g of Na2SeO4, 0.003-0.005g of Na2WO4, in 1L of deionized water and store at 4-6℃.

[0012] Preferably, the concentrated compound vitamin solution contains: 1.0-3.0 mg biotin, 1.0-3.0 mg folic acid, 8.0-12.0 mg vitamin B6, 4.0-6.0 mg vitamin B1, and vitamin B... 12 Dissolve 0.008-0.012 mg of riboflavin, 4.0-6.0 mg of niacin, 4.0-6.0 mg of calcium pantothenate, and 0.8-1.2 mg of para-aminobenzoic acid in 1 L of deionized water and store at 4-6 °C.

[0013] In this invention, Citrobacter freundii ( Citrobacter freundii Biological preservation information for YGgY-138:

[0014] Date of deposit: September 16, 2022;

[0015] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0016] Address of depositary institution: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Accession number: CGMCC No. 25749, Institute of Microbiology, Chinese Academy of Sciences;

[0017] Classification and nomenclature: Citrobacter freundii ( Citrobacter freundii ).

[0018] Advantages of this invention:

[0019] The present invention provides Citrobacter freundii ( Citrobacter freundii This strain is a microorganism that can improve the bioconversion of CO2 to methane. It can still grow actively in the reservoir environment and convert CO2 into CH4. It has great application potential in the fields of reservoir CO2 storage and bioconversion to CH4. Attached Figure Description

[0020] Figure 1 Phylogenetic tree of 16S rDNA of strain YGgY-138;

[0021] Figure 2 The relationship between the biomass and CH4 production activity of strain YGgY-138;

[0022] Figure 3 To simulate the change in microbial biomass over time in a reaction system under reservoir conditions;

[0023] Figure 4 To simulate the change of CO2 in the reaction system over time under reservoir conditions;

[0024] Figure 5 To simulate the change of CH4 in the reaction system over time under reservoir conditions. Detailed Implementation

[0025] Example 1

[0026] I. The culture medium used in this invention

[0027] 1. Enrichment medium: Take 0.4g K2HPO4·3H2O, 0.2g KH2PO4, 1.0g NH4Cl, 0.1g MgCl2·6H2O, 2.0g sodium acetate, 2.0g sodium formate, 1.0g yeast extract, 1.0g tryptone, 0.5g L-cysteine ​​hydrochloride, and 1ml 0.1% resazurite. Dissolve all the above reagents in 1L of deionized water. Adjust the pH to 7.0 with 1mol / L hydrochloric acid, boil to remove oxygen, and autoclave at 121℃ for 15 minutes. Before use, add 1.0mL of trace element concentrate, 1.0mL of compound vitamin concentrate, 20mL of 1% Na2S·9H2O, and 10mL of 10% NaHCO3. All four solutions must be filtered through a sterile filter before addition.

[0028] 2. Solid separation culture medium: Take 0.4g K2HPO4·3H2O, 0.2g KH2PO4, 1.0g NH4Cl, 0.1g MgCl2·6H2O, 2.0g sodium acetate, 2.0g sodium formate, 1.0g yeast extract, 1.0g tryptone, 0.5g L-cysteine ​​hydrochloride, 1mL 0.1% resazurite, and 15g agar. Dissolve all the above reagents in 1L of deionized water. Adjust the pH to 7.0 with 1mol / L hydrochloric acid, boil to remove oxygen, and autoclave at 121℃ for 15 minutes. When the temperature drops below 60℃, add 1.0mL of trace element concentrate, 1.0mL of compound vitamin concentrate, 20mL of 1% Na2S·9H2O, and 10mL of 10% NaHCO3 sequentially through a sterile filter, mix well, and dispense into anaerobic tubes.

[0029] 3. Trace element concentrate: Dissolve 12.0g of aminotriacetic acid (NTA), 1.25g of FeCl3·6H2O, 0.024g of CoCl2·6H2O, 0.1g of CaCl2·2H2O, 0.1g of MnCl2·4H2O, 0.1g of ZnCl2, 0.1g of CuCl2·2H2O, 0.025g of H3BO3, 0.1g of Na2MoO4·2H2O, 1.0g of NaCl, 0.12g of NiCl2·6H2O, 0.004g of Na2SeO4, and 0.004g of Na2WO4 in 1L of deionized water sequentially, and store at 4℃ for later use.

[0030] 4. Concentrated Multivitamin Solution: Contains 2.0mg Biotin, 2.0mg Folic Acid, 10.0mg Vitamin B6, 5.0mg Vitamin B1, and 10.0mg Vitamin B2. 12 Dissolve 0.01 mg of riboflavin, 5.0 mg of niacin, 5.0 mg of calcium pantothenate, and 1.0 mg of para-aminobenzoic acid in 1 L of deionized water and store at 4 °C for later use.

[0031] II. Citrobacter freundii ( Citrobacter freundii Screening of strains

[0032] 1. Enrichment of bacterial strains

[0033] 10 mL of produced fluid from the Xingzichuan Oil Production Plant of Yanchang Oilfield, stored at 4℃, was inoculated into an anaerobic culture bottle containing 90 mL of enrichment medium using a sterile syringe in a sterile anaerobic operating box. A mixed gas (N2:H2:CO2=85%:10%:5%) was added, the bottle was quickly sealed, and the bottle was placed in an anaerobic incubator at 30℃ for enrichment culture for 7 days. Then, 10 mL of the culture solution was taken and inoculated again into a new anaerobic culture bottle containing 90 mL of enrichment medium for 7 days. The above operation was repeated 3 times.

[0034] 2. Isolation of single strains

[0035] The Hungate roll tube method was used to isolate single strains. First, the bacterial suspension was diluted 10,000 times using an anaerobic Hungate method, and then inoculated into an anaerobic test tube containing melted solid isolation medium using a syringe. The test tube was tightly sealed with a butyl rubber stopper and placed horizontally in an ice bath, where it was rolled evenly to ensure the bacterial medium covered the entire inner surface of the tube. The tube was then incubated at 30°C for 7 days. After colonies appeared, the isolation and culture process was repeated until a single strain was completely isolated and named YGgY-138. This strain was then stored at 4°C in an anaerobic tube for later use.

[0036] III. Identification of a single strain YGgY-138

[0037] The species of the isolated strain was determined by comparing its 16S rDNA sequence. The genome of strain YGgY-138 was extracted using the PowerSoil DNA Isolation Kit, and the 16S rDNA sequence was amplified by PCR using the following primers as a template:

[0038] Upstream primer (F27): 5'-AGAGTTTGATCCTGGCTCAG-3',

[0039] Downstream primer (R1492): 5'-TACGGCTACCTTGTTACGACTT-3',

[0040] The PCR reaction system is as follows:

[0041] 2×Pfu Master Mix 25μL

[0042] Upstream primer 2μL,

[0043] 2 μL of downstream primer

[0044] Template, 2μL

[0045] Add ddH2O to 50 μL;

[0046] 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.

[0047] 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. Figure 1 The strain YGgY-138 was ultimately identified as belonging to *Citrobacter freundii*. Citrobacter freundii ) genus, named Citrobacter freundii YGgY-138.

[0048] The strain YGgY-138 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. 25749, and classified as *Citrobacter freundii*. Citrobacter freundii ).

[0049] IV. CH4 production bioactivity of strain YGgY-138

[0050] Anaerobic incubator was used for anaerobic operation. The incubator was first purged three times with 99.999% high-purity N2, then purged again with an H2 / CO2 (V / V = 4:1) mixture, maintaining the H2 / CO2 ratio throughout. Single bacteria were picked from anaerobic tubes (YGgY-138 stored at 4℃) and inoculated into 250mL Erlenmeyer flasks containing 100mL of enrichment medium. The flasks were tightly capped and cultured at 30℃ in the anaerobic incubator until the logarithmic growth phase, yielding a seed culture of *Citrobacter freundii*. This seed culture was then inoculated into new enrichment medium at inoculation rates of 0%, 5%, 10%, and 15% (v / v), and cultured for 10 days at 30℃ using an H2 / CO2 (V / V = 4:1) mixture as the reaction substrate. After culturing, the gas in the reaction flasks was collected using a gas collecting tube, and the methane content was determined by gas chromatography. The methane content was calculated as the equivalent H2 / CO2 ratio per 100mL of medium. CO2 (V / V=4 : 1) is 150mL;

[0051] The CH4 production was investigated when the inoculum concentration of Citrobacter freundii was 0%, 5%, 10%, and 15%. Figure 2 It was found that as the inoculum size of *Citrobacter freundii* increased, the final methane production of the system gradually increased, especially at an inoculum size of 10%, where the methane production increased rapidly, and the CH4 production at the end of the reaction reached 5.22 mL. When the inoculum size was 15%, the CH4 production of the reaction system continued to increase, reaching 6.15 mL, but the increase was not significant. This indicates that when the inoculum size is greater than or equal to 15%, due to the limited concentration of nutrients in the system, the proliferation of *Citrobacter freundii* slows down after reaching a certain concentration, i.e., it reaches a stationary phase. Therefore, the optimal inoculum size for the reaction system is 10%~15% v / v, which yields a higher CH4 production.

[0052] V. High-Pressure Model Experiment of CO2 Bioconversion to CH4 in Oil Reservoir mediated by Citrobacter freundii

[0053] raw material:

[0054] After filtering the produced fluid from the oil well and separating the oil and water, the water sample was mixed with the dehydrated crude oil at a volume ratio of 3:1 to obtain an oil-water mixture sample.

[0055] Rocks and minerals: samples were taken from the Chang 2 reservoir of Xingzichuan Oil Production Plant in Yanchang Oilfield. After crushing and sieving, rock samples with a particle size of 1-2 mm were selected as rocks and minerals.

[0056] Each liter of the oil-water mixture contains the following nutrients: 0.4g K₂HPO₄·3H₂O, 0.2g KH₂PO₄, 1.0g NH₄Cl, 0.1g MgCl₂·6H₂O, 2.0g sodium acetate, 1.0g sodium formate, 1.0g yeast extract, 1.0g tryptone, 0.5g L-cysteine ​​hydrochloride, 1.0mL 0.1% resazurite, 1.0mL concentrated trace elements, 1.0mL concentrated compound vitamins, 20mL 1% Na₂S·9H₂O, and 10mL 10% NaHCO₃.

[0057] The experiment is as follows:

[0058] 360 mL of oil-water mixture, nutrients, 40 mL of *Citrobacter freundii* seed culture, and 40 g of rock minerals were added to a 500 mL high-pressure reactor (solid-liquid ratio 1:10). Simultaneously, two blank controls were set up: 1) with 400 mL of oil-water mixture and 40 g of rock minerals added to the same reactor; 2) with 400 mL of oil-water mixture, nutrients, and 40 g of rock minerals added to the same reactor. Then, 140 mL of 99.9% CO2 fluid was pressurized to 8 MPa using a booster pump and introduced into the reactor. The reactor temperature was adjusted to 28–30 °C using an external magnetically stirred heating device. The reaction time was at least 30 days. During the reaction period, 5 mL of the reaction solution was collected on days 7, 14, 21, and 30 to measure its OD value. 600 The value reflects the biomass of bacteria in the reaction solution. Figure 3 Simultaneously, headspace gas samples were taken to determine the CO2 and CH4 content in the gas, and the amount of CO2 consumed and CH4 produced by the system was calculated. Figure 4 , Figure 5 ).

[0059] Depend on Figure 3 It can be seen that the Citrobacter freundii provided by the present invention ( Citrobacter freundii Strain YGgY-138 was subjected to high-pressure physical model tests with rocks, minerals, oil-water mixtures, and CO2 fluids. The results showed that when a certain amount of nutrient was added to the reaction system, the biomass of *Citrobacter freundii* increased rapidly over time, reaching an OD value of [missing value] on day 30. 600 The biomass of control 1 (without nutrients and Citrobacter freundii seed culture) and control 2 (with only nutrients) increased more slowly. The OD of control 1 was 0.75. 600 The OD value was only 0.32, while the blank control 2, due to the addition of nutrients, activated the methanogenic bacteria originally present in the water sample, thus resulting in a higher OD value. 600 The biomass reached 0.5, but compared to the experimental group which had added Citrobacter freundii seed liquid, the increase in biomass in both control groups was much lower than that in the experimental group.

[0060] Depend on Figure 4 and Figure 5 It can be seen that the Citrobacter freundii provided by the present invention ( Citrobacter freundii Strain YGgY-138 was subjected to high-pressure physical model tests with rocks, minerals, oil-water mixtures, and CO2 fluids. The results showed that, mediated by Citrobacter freundii, the reaction system consumed a total of 95.71 mL of CO2 on day 30, with an average CO2 consumption rate of 3.19 mL / d. At the same time, the total amount of CH4 produced by the reaction system on day 30 was 8.68 mL, and the methane production rate was 0.289 mL / d. Compared with the blank control 1, the CO2 consumption rate in the system increased by 3.1 times, and the methane production rate increased by 4.5 times.

[0061] Therefore, it can be seen that the *Citrobacter freundii* of the present invention can still grow actively in the reservoir environment and convert CO2 into CH4 through a series of biochemical reactions and reservoir chemical reactions. In the reservoir environment, it can significantly increase the rate of CO2 to methane conversion and has great application potential in the fields of reservoir CO2 storage and bioconversion to CH4.

[0062] Example 2

[0063] Citrobacter freundii ( Citrobacter freundii In the application of CO2 bioconversion to methanogenesis in oil reservoirs, the reservoirs are capable of CO2 flooding or sequestration:

[0064] As can be seen from the high-pressure model test in Example 1 above, *Citrobacter freundii* (… Citrobacter freundii In reservoir environments, the rate of CO2 conversion to methane can be significantly increased. Therefore, the *Citrobacter freundii* (…) Citrobacter freundii The application of *Citrobacter freundii* (Citrobacter freundii) to methanogenesis in oil reservoirs is as follows: During CO2 flooding or sequestration, while injecting CO2 into the reservoir, *Citrobacter freundii* is also introduced into the reservoir. Citrobacter freundii The seed solution was diluted with water to 5-10 wt% and then injected into the oil reservoir.

[0065] The Citrobacter freundii ( Citrobacter freundiiThe seed culture of *Citrobacter freundii* was prepared as follows: Anaerobic operation was performed using an anaerobic incubator. The incubator was first purged three times with 99.999% high-purity N2, then purged again with a H2 / CO2 (V / V=4:1) mixture, maintaining the H2 / CO2 ratio throughout. Single bacteria were picked from anaerobic tubes (YGgY-138 stored at 4-6℃) and inoculated into 250mL Erlenmeyer flasks containing 100mL of enrichment medium. The flasks were tightly capped, and cultured in an anaerobic incubator at 28-32℃ until the logarithmic growth phase using a H2+CO2 (V / V=4 / 1) mixture as both electron donor and energy substrate, yielding the seed culture of *Citrobacter freundii*.

[0066] The enrichment medium per liter of reaction solution is formulated as follows: K₂HPO₄·3H₂O 0.3-0.5 g, KH₂PO₄ 0.1-0.3 g, NH₄Cl 0.8-1.2 g, MgCl₂·6H₂O 0.08-0.12 g, sodium acetate 1.5-2.5 g, sodium formate 1.5-2.5 g, yeast extract 0.8-1.2 g, tryptone 0.8-1.2 g, L-cysteine ​​hydrochloride 0.4-0.6 g, 0.1% resazurite 0.8-1.2 mL, and deionized water 1 L. This solution is boiled to remove oxygen and then autoclaved at 121℃ for 15 minutes. Before use, add 0.8-1.2 mL of concentrated trace element solution, 0.8-1.2 mL of concentrated compound vitamin solution, and 1% Na₂S·2H₂O. 18-22 mL of 9H2O and 8-12 mL of 10% NaHCO3. All four solutions must be filtered through a sterile filter before addition.

[0067] Preferably, after dilution, nutrients are added before injection into the reservoir; wherein each liter of injected water contains the following nutrients: K2HPO4·3H2O 0.3-0.5g, KH2PO4 0.1-0.3g, NH4Cl 0.8-1.2g, MgCl2·6H2O 0.08-0.12g, sodium acetate 1.5-2.5g, sodium formate 1.5-2.5g, yeast extract 0.8-1.2g, tryptone 0.8-1.2g, L-cysteine ​​hydrochloride 0.4-0.6g, 0.1% resazurite 0.8-1.2mL, trace element concentrate 0.8-1.2mL, compound vitamin concentrate 0.8-1.2mL, 1% Na2S·9H2O 18-22mL, and 10% NaHCO3 8-12mL.

[0068] The trace element concentrate is as follows: 10.0-14.0 g of aminotriacetic acid, 1.2-1.5 g of FeCl3·6H2O, 0.020-0.026 g of CoCl2·6H2O, 0.08-0.12 g of CaCl2·2H2O, 0.08-0.12 g of MnCl2·4H2O, 0.08-0.12 g of ZnCl2, 0.020-0.030 g of CuCl2·2H2O, 0.08-0.12 g of H3BO3, 0.022-0.026 g of Na2MoO4·2H2O, 0.8-1.2 g of NaCl, and NiCl2·6H2O. Dissolve 0.10-0.14g of Na₂SeO₄, 0.003-0.005g of Na₂WO₄, in 1L of deionized water and store at 4-6℃.

[0069] The concentrated multivitamin solution contains: biotin 1.0-3.0 mg, folic acid 1.0-3.0 mg, vitamin B6 8.0-12.0 mg, vitamin B1 4.0-6.0 mg, and vitamin B... 12 Dissolve 0.008-0.012 mg of riboflavin, 4.0-6.0 mg of niacin, 4.0-6.0 mg of calcium pantothenate, and 0.8-1.2 mg of para-aminobenzoic acid in 1 L of deionized water and store at 4-6 °C.

Claims

1. A type of Citrobacter freundii that produces methanates from carbon dioxide bioconversion in oil reservoirs ( Citrobacter freundii ), characterized in that: It is Citrobacter freundii (Citrobacter freundii Strain YGgY-138 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 16, 2022, with accession number CGMCCNo.25749.

2. The Citrobacter freundii as described in claim 1 ( Citrobacter freundii Its application in the bioconversion of CO2 in oil reservoirs to produce methanogens is characterized by: The reservoir is capable of CO2 flooding or sequestration; the application involves injecting CO2 into the reservoir during CO2 flooding or sequestration while simultaneously injecting the *Citrobacter freundii* (…). Citrobacter freundii The seed solution was diluted with water to 5-10 wt% and then injected into the oil reservoir.

3. The Citrobacter freundii according to claim 2 ( Citrobacter freundii Its application in the bioconversion of CO2 in oil reservoirs to produce methanogens is characterized by: After dilution, nutrients need to be added before injecting into the reservoir. Each liter of injected water contains the following nutrients: K₂HPO₄·3H₂O 0.3-0.5g, KH₂PO₄ 0.1-0.3g, NH₄Cl 0.8-1.2g, MgCl₂·6H₂O 0.08-0.12g, sodium acetate 1.5-2.5g, sodium formate 1.5-2.5g, yeast extract 0.8-1.2g, tryptone 0.8-1.2g, L-cysteine ​​hydrochloride 0.4-0.6g, 0.1% resazurite 0.8-1.2mL, trace element concentrate 0.8-1.2mL, compound vitamin concentrate 0.8-1.2mL, 1% Na₂S·9H₂O 18-22mL, and 10% NaHCO₃ 8-12mL.

4. The Citrobacter freundii according to claim 3 ( Citrobacter freundii Its application in the bioconversion of CO2 in oil reservoirs to produce methanogens is characterized by: The trace element concentrate is as follows: 10.0-14.0g of aminotriacetic acid, 1.2-1.5g of FeCl3·6H2O, 0.020-0.026g of CoCl2·6H2O, 0.08-0.12g of CaCl2·2H2O, 0.08-0.12g of MnCl2·4H2O, 0.08-0.12g of ZnCl2, 0.020-0.030g of CuCl2·2H2O, 0.08-0.12g of H3BO3, 0.022-0.026g of Na2MoO4·2H2O, 0.8-1.2g of NaCl, and NiCl2·6H2O. Dissolve 0.10-0.14g of Na₂SeO₄, 0.003-0.005g of Na₂WO₄, and 0.003-0.005g of Na₂WO₄ in 1L of deionized water and store at 4-6℃.

5. The Citrobacter freundii according to claim 3 ( Citrobacter freundii Its application in the bioconversion of CO2 in oil reservoirs to produce methanogens is characterized by: The concentrated multivitamin solution contains: biotin 1.0-3.0 mg, folic acid 1.0-3.0 mg, vitamin B6 8.0-12.0 mg, vitamin B1 4.0-6.0 mg, and vitamin B... 12 Dissolve 0.008-0.012 mg of riboflavin, 4.0-6.0 mg of niacin, 4.0-6.0 mg of calcium pantothenate, and 0.8-1.2 mg of para-aminobenzoic acid in 1 L of deionized water and store at 4-6 °C.

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