Rhizobium, polysaccharide biopolymer produced thereby, and use thereof

Abstract

This invention discloses a rhizobium, the polysaccharide biopolymer produced by it, and their applications. The rhizobium is classified as Rhizobium (…). Rhizobium sp.) L001, with accession number CCTCC NO: M20222005. This rhizobium can ferment to produce polysaccharide biopolymers. The functional component of the produced polysaccharide biopolymer is a novel extracellular polysaccharide, mainly composed of glucose residues and galactose residues in a molar ratio of 3:1. This polysaccharide biopolymer exhibits shear-thinning properties, undergoes phase inversion at different concentrations, and displays gel-like characteristics. It also possesses advantages such as high viscosity, emulsifying activity, high temperature resistance, strong salt tolerance, and a wide applicable pH range. In particular, it can withstand high temperature and high salt conditions, especially under extreme conditions of pH 1 or pH 12, showing promising application prospects.

Description

A rhizobium, its produced polysaccharide biopolymers and their applications Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a rhizobium, the polysaccharide biopolymer produced by it, and its applications. Background Technology

[0002] Polymer flooding is one of the most promising technologies for enhancing the productivity of mature oil reservoirs. Polymers reduce the injection water-to-crude oil mobility ratio, improving the displacement of trapped oil and thus increasing oil recovery. With the deepening of oilfield development, most of my country's oilfields have been exploited, leaving harsh reservoirs with high temperatures and high salinity that are difficult to displace using chemical polymers. Polyacrylamide (HPAM), a commonly used chemical polymer in tertiary oil recovery, has limited effectiveness under high temperature and high salinity conditions and degrades into toxic acrylamide monomers, causing environmental pollution and accumulating in the human body, posing a health risk. Therefore, it is necessary to develop more environmentally friendly and efficient biopolymers to replace HPAM. Currently, biopolymers such as xanthan gum and vesico-laminar gum have already been applied in the oil extraction field.

[0003] Among bacteria known to synthesize extracellular polysaccharides, those synthesized by *Rhizobium* exhibit rich structural diversity. Rhizobium extracellular polysaccharides are heterogeneous microbial polysaccharides secreted by *Rhizobium* microorganisms during their growth process, composed of a certain number of repeating units. Biopolymers produced by *Rhizobium CECT 908* exhibit better rheological properties than xanthan gum, especially at high shear rates (up to 300 s⁻¹). -1 It maintains high stability under various conditions, including high temperature (up to 80°C) and salinity (up to 200 g / L NaCl), but there is currently no research to prove that rhizobium polysaccharides are suitable for harsh oil reservoirs with relatively extreme pH. Summary of the Invention

[0004] The first objective of this invention is to provide a rhizobium, which is classified as Rhizobium sp. L001 and has the accession number CCTCC NO: M20222005.

[0005] A second objective of this invention is to provide the application of the above-mentioned rhizobium in the production of polysaccharide biopolymers.

[0006] In one preferred embodiment, the rhizobium is fermented to obtain polysaccharide biopolymers.

[0007] A third objective of the present invention is to provide a polysaccharide biopolymer obtained by producing the rhizobium as described in claim 1.

[0008] In a preferred embodiment, the main components of the polysaccharide biopolymer are galactose and glucose in a molar ratio of 1:3.

[0009] A fourth objective of this invention is to provide the application of the above-mentioned polysaccharide biopolymers in the preparation of bioemulsifiers.

[0010] This polysaccharide biopolymer is used as a bioemulsifier in the form of fermentation broth or biopolymer solution.

[0011] Preferably, the mass concentration of the polysaccharide biopolymer in the biopolymer solution is higher than 0.5%. When the mass concentration of the polysaccharide biopolymer is 0.5%, the emulsification index of n-hexane can reach more than 50%. When the concentration reaches 1% or above, the emulsion layer structure formed is compact and will not collapse even when inverted, indicating that the polysaccharide biopolymer has good emulsifying properties and can be used as a bio-emulsifier.

[0012] The fifth objective of this invention is to provide the application of the above-mentioned polysaccharide biopolymers in polymer flooding.

[0013] In a preferred embodiment, the polysaccharide biopolymer is used in the form of fermentation broth or biopolymer solution for biopolymer flooding in oil reservoirs.

[0014] In a preferred embodiment, the fermentation broth is obtained by fermenting rhizobia, and the fermentation broth is diluted to a preset mass concentration of polysaccharides before use.

[0015] The preparation method of the biopolymer solution is as follows: extract extracellular polysaccharides from the fermentation broth, completely swell the extracellular polysaccharides, and dilute with water until the polysaccharide content reaches a preset mass concentration, which is the biopolymer solution.

[0016] In one preferred embodiment, the polysaccharide biopolymer is used in reservoir environments with pH=1-12; preferably in reservoir environments with pH=1-3 or pH=11-12.

[0017] In a preferred embodiment, the polysaccharide biopolymer is used in reservoir environments with temperatures ≤60°C.

[0018] The Rhizobium L001 strain of this invention can produce high levels of extracellular polysaccharides, reaching 22.8 g / L (pure extracellular polysaccharide) under shake-flask conditions. The extracellular polysaccharide produced by this strain is mainly composed of glucose and galactose residues in a molar ratio of 3:1, which is distinct from the extracellular polysaccharides produced by existing Rhizobium strains, representing a novel extracellular polysaccharide product. The polysaccharide biopolymers containing this extracellular polysaccharide exhibit shear-thinning properties, undergo phase inversion at different concentrations, possess gel properties, and have advantages such as high viscosity, high temperature resistance, strong salt resistance, and a wide applicable pH range. Especially under extreme conditions of pH 1 or pH 12, it can withstand high temperature and high salt, possessing shear-thinning properties and temperature and salt resistance suitable for the petroleum industry, showing excellent application potential. Furthermore, the excellent performance of the polysaccharide polymers produced by this strain in extremely acidic environments makes it a promising thickener for acid fracturing. Attached Figure Description

[0019] Figure 1 shows the colony morphology of Rhizobium sp. L001.

[0020] Figure 2 is an ion chromatogram of the extracellular polysaccharide produced by Rhizobium sp. L001.

[0021] Figure 3 shows the shear rate of extracellular polysaccharides produced by Rhizobium sp. L001.

[0022] Figure 4 is a frequency scan of the extracellular polysaccharides produced by Rhizobium sp. L001.

[0023] Figure 5 is an oscillatory strain diagram of the extracellular polysaccharide produced by Rhizobium sp. L001.

[0024] Figure 6 shows the emulsifying properties of extracellular polysaccharides produced by different concentrations of Rhizobium sp. L001 on n-hexane.

[0025] Figure 7 shows the emulsifying properties of the extracellular polysaccharide produced by high-concentration Rhizobium sp. L001 on n-hexane.

[0026] Figure 8 is a temperature-viscosity diagram of the extracellular polysaccharide produced by Rhizobium sp. L001.

[0027] Figure 9 is a mineralization-viscosity diagram of extracellular polysaccharides produced by Rhizobium sp. L001.

[0028] Figure 10 is a pH-viscosity diagram of extracellular polysaccharides produced by Rhizobium sp. L001.

[0029] Figure 11 shows the temperature-viscosity graph of the extracellular polysaccharide produced by Rhizobium sp. L001 at different pH values ​​when the mineralization is 5000 mg / L.

[0030] Figure 12 shows the temperature-viscosity graph of the extracellular polysaccharide produced by Rhizobium sp. L001 at different pH values ​​when the mineralization is 20000 mg / L.

[0031] Figure 13 shows the temperature-viscosity graph of the extracellular polysaccharide produced by Rhizobium sp. L001 at different pH values ​​when the mineralization is 50,000 mg / L.

[0032] Figure 14 shows the temperature-viscosity graph of the extracellular polysaccharide produced by Rhizobium sp. L001 in an oil-containing environment at different pH values ​​when the mineralization is 50,000 mg / L.

[0033] The biological material described in this invention, classified as Rhizobium sp. L001, was deposited on December 23, 2022, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20222005, and deposit address: Wuhan, China. Detailed Implementation Example 1

[0034] This embodiment illustrates the isolation and identification method and results of Rhizobium sp. L001.

[0035] A colorless, smooth-surfaced bacterial strain was isolated from the root soil of tropical plants using the 96-well plate method. It grew well on sugar-containing medium, forming raised, smooth, and sticky colonies (Figure 1). The colonies were easily drawn into strings when picked. The strain was then cultured and identified.

[0036] The steps include:

[0037] (1) Extraction of total bacterial genomic DNA: Take 200 μL of Hizobium sp. L001 bacterial culture from the bacterial culture preservation tube and put it into LB medium. Incubate at 30℃ and 200 rpm for 24 h. Extract total DNA using a genomic DNA extraction kit.

[0038] (2) PCR amplification of bacterial 16S rDNA: The amplification products were detected by 1% agarose gel electrophoresis using the universal bacterial primers 27F and 1492R, and sent to a sequencing company for sequencing identification.

[0039] Reaction system: ddH2O: 20 μL; high-fidelity enzyme mixture: 25 μL; primer 1: 2 μL; primer 2: 2 μL; template: 1 μL

[0040] Reaction conditions: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 30 s / 1 kb, 30 cycles followed by 72℃ extension for 5 min.

[0041] The sequencing results of the partial 16S rDNA sequence are shown in SEQ ID NO: 1. After comparison using NCBI BLAST analysis software, it was shown that the 16S rDNA gene sequence of this strain shares more than 99% homology with that of the tropical rhizobium (Rhizobium tropici) strain. Therefore, the L001 strain was identified as belonging to the genus Rhizobium and named Rhizobium sp. L001. Example 2

[0042] This embodiment illustrates a method for cultivating extracellular polymeric substances from Rhizobium sp. L001 (CCTCC NO: M20222005), the steps of which include:

[0043] (1) Take 200 μL of Hizobium sp. L001 bacterial solution from the bacterial culture tube into an Erlenmeyer flask containing bacterial activation medium, and incubate at 30℃ and 200 rpm for 24 h. Use an inoculation loop to take a small amount of bacterial solution and streak it on the bacterial activation plate, and incubate at 30℃ for 48 h.

[0044] (2) Use an inoculation loop to pick up a single colony with good growth on the activation plate and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium. Place the flask in a shaker at 30℃ and 200 rpm for 24 h.

[0045] (3) Inoculate the cultured seed liquid into a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium at an inoculation rate of 6% (v / v) and place it in a shaker at 30℃ and 200 rpm for 72 h.

[0046] Microbial activation medium: 5 g peptone, 3 g beef extract, 5 g NaCl, 15 g agar, 1 L water, sterilized at 121℃ for 20 min.

[0047] Seed culture medium: 20 g sucrose, 1 g yeast extract, 4 g peptone, 2 g K2HPO4, 0.1 g MgSO4, 1 L water, pH 7.0-7.2, sterilized at 121℃ for 20 min.

[0048] Fermentation medium: 40 g sucrose, 1 g yeast extract, 4 g peptone, 2 g K₂HPO₄, 0.1 g MgSO₄, 1 L water, pH 7.0-7.2, sterilized at 121℃ for 20 min. Example 3

[0049] This embodiment illustrates a method for extracting extracellular polysaccharides from Rhizobium sp. L001, the steps of which include:

[0050] (1) Extraction of crude extracellular polysaccharide: The fermentation broth in Example 2 was placed in an 80°C water bath for 20 min, diluted with an equal volume of distilled water, centrifuged at 8000 r / min for 30 min to remove bacteria, the supernatant was collected, concentrated and then 3 times the volume of 95% ethanol was added, mixed well, placed in a 4°C refrigerator and left to stand overnight, centrifuged at 8000 r / min for 30 min to remove the supernatant, repeated several times, the precipitate was collected, and dried in an 80°C oven to constant weight to obtain crude extracellular polysaccharide.

[0051] (2) Protein removal: Dissolve an appropriate amount of crude product in 100 mL of distilled water, heat and stir until dissolved, add 20 mL of chloroform: n-butanol = 4:1 solution, shake for 30 min, centrifuge at 8000 r / min for 30 min and collect the supernatant. Repeat several times until no oily substance appears in the organic phase.

[0052] (3) Freeze-drying: The crude product after protein removal is placed in a dialysis bag with a molecular weight cutoff of 10,000 Da, concentrated with polyethylene glycol, dialyzed for 3 days, and the dialysis solution is freeze-dried to obtain pure extracellular polysaccharide.

[0053] After polysaccharide extraction from the fermentation broth of Rhizobium sp. L001 in Example 2, the yield of pure extracellular polysaccharides reached 22.8 g / L. Example 4

[0054] This example illustrates the monosaccharide composition analysis results of the extracellular polysaccharides produced by Rhizobium sp. L001.

[0055] Take 10 mg of the pure L001 polysaccharide extracted in Example 3 and place it in an ampoule. Add 10 mL of 3M TFA and hydrolyze at 120 °C for 3 h. Accurately pipette the acid hydrolysis solution into a tube and blow it dry under nitrogen. Add 5 mL of water and vortex to mix. Pipette 100 μL of the solution into 900 μL of deionized water and centrifuge at 12000 rpm for 5 min. Take the supernatant for ion chromatography analysis.

[0056] Figure 2 shows the ion chromatogram of the extracellular polysaccharide hydrolysis system of Rhizobium sp. L001. The main hydrolysis product peaks of the L001 polysaccharide component are galactose (14.617 min) and glucose (16.592 min), with a molar ratio of approximately 1:3. This result indicates that the extracellular polysaccharide produced by Rhizobium sp. L001 is composed of glucose residues and galactose residues in a 3:1 ratio.

[0057] Currently, the extracellular polysaccharides synthesized in large quantities by reported Rhizobium spp. strains commonly contain glucose, galactose, galacturonic acid, rhamnose, and mannose. For example, the extracellular polysaccharide of *Rhizobium peaense* consists of glucose residues, glucuronic acid residues, and galactose residues in a 5:2:1 ratio; the extracellular polysaccharide produced by *Rhizobium sinense* of alfalfa consists of glucose residues and galactose residues in a 7:1 ratio; the extracellular polysaccharide of *Rhizobium esculentum* of soybean consists of glucose residues, mannose residues, galactose residues, and galacturonic acid residues in a 2:1:1:1 ratio; and the extracellular polysaccharide of *Rhizobium esculentum* consists of rhamnose residues and glucuronic acid residues in a 5:1 ratio. The extracellular polysaccharide component produced by Rhizobium sp. L001 is similar to that produced by Rhizobium sinense from alfalfa, both consisting of glucose and galactose residues. However, the proportion of monosaccharide residues differs significantly. Therefore, L001 polysaccharide is a novel rhizobium polysaccharide. Example 5

[0058] This example illustrates the physicochemical analysis results of the extracellular polysaccharide produced by Rhizobium sp. L001.

[0059] A small amount of pure L001 polysaccharide extracted in Example 3 was taken for physicochemical analysis, and the component content was calculated by preparing a corresponding standard curve.

[0060] The total sugar content in the polysaccharide sample, determined by the phenol-sulfuric acid method, was 83.03%, and the protein content, determined by the Coomassie brilliant blue method, was 0.68%. No uronic acid components were detected in the L001 polysaccharide sample using the carbazole-sulfuric acid method. This result is consistent with the results of Example 4, indicating that uronic acid components are not present in the L001 polysaccharide. Example 6

[0061] This example illustrates the rheological properties of extracellular polysaccharides produced by Rhizobium sp. L001.

[0062] (1) Preparation of polysaccharide polymer solution: Weigh a certain mass of the biopolymer powder prepared in Example 3, dissolve it in an appropriate amount of water, stir at low speed to make it swell completely, and prepare L001 polysaccharide polymer solutions with mass concentrations of 0.1%, 0.25%, 0.5%, 0.75% and 1.0% respectively.

[0063] (2) Effect of shear rate on polymer viscosity: At room temperature, the viscosity of L001 polysaccharide polymer solutions with mass concentrations of 0.1%, 0.25%, 0.5%, 0.75% and 1.0% was measured at different shear rates using a rotational rheometer. The results are shown in Figure 3. The viscosity of the L001 polysaccharide polymer aqueous solution increased with increasing concentration. At the same time, it was found that the viscosity decreased sharply and tended to level off as the shear rate continued to increase, showing typical pseudoplastic fluid characteristics—the apparent viscosity of the fluid decreased with increasing shear rate, i.e., shear thinning phenomenon.

[0064] (3) Effect of frequency variation on polymer morphology: At room temperature, the modulus of L001 polysaccharide polymer solutions with mass concentrations of 0.1%, 0.25%, 0.5%, 0.75%, and 1.0% were measured at different frequencies using a rotational rheometer. The results are shown in Figure 4. L001 polysaccharide polymer underwent phase inversion at all concentrations, changing from a molten state to a gel state, i.e., from a fluid-like state to a solid-like state. This intersection point is the gel point, exhibiting typical behavior characteristics of a non-crosslinked polymer. However, as the concentration increases, the elastic modulus and viscous modulus also increase, and the intersection point shifts to the left, indicating that the molecular weight distribution width is narrower and the average molecular weight is larger at higher concentrations.

[0065] (4) Effect of stress change on polymer morphology: At room temperature, the modulus of L001 polysaccharide polymer solutions with mass concentrations of 0.5%, 0.75%, and 1.0% under different strains was measured using a rotational rheometer. The results are shown in Figure 5. At the low concentration (0.5%), the viscous modulus was comparable to the elastic modulus. As the concentration increased, the elastic modulus became greater than the viscous modulus, and there were intersections at each concentration, indicating that phase inversion occurred and the polysaccharide morphology changed from a gel state to a melt state. The rightward shift of the intersection also indicates that the degree of intermolecular entanglement changed. Example 7

[0066] This example illustrates the evaluation of the emulsifying properties of the polysaccharide polymer from Rhizobium sp. L001.

[0067] (1) Preparation of polysaccharide polymer solution: Weigh a certain mass of the biopolymer powder prepared in Example 3, dissolve it in an appropriate amount of water, stir at low speed to make it swell completely, and prepare L001 polysaccharide polymer solutions with mass concentrations of 0.25%, 0.5%, 0.75%, 1.0% and 1.5% respectively.

[0068] (2) Determination of emulsification index: Add organic phase and L001 polysaccharide solution to test tubes according to the ratio, use a turbine shaker to shake at 3000 r / min for 2 min, let stand for 24 h, measure the height of the emulsion layer and the total height of the liquid surface, and calculate EI. 24 .

[0069]

[0070] (3) Emulsifying properties of L001 polysaccharide polymers with different concentrations on n-hexane: With n-hexane as the organic phase, the emulsification index can still reach over 50% when the concentration of the extracellular polysaccharide solution is as low as 0.5% (Figure 6). When the concentration reaches 1% or above, the emulsion layer structure is compact and will not collapse even when inverted (Figure 7). This indicates that L001 polysaccharide has good emulsifying properties and can be used as a bio-emulsifier to stabilize the emulsion by increasing kinetic stability, thus enabling its application in the petroleum field. Example 8

[0071] This example illustrates the evaluation of the temperature and salt resistance properties of the polysaccharide polymer of Rhizobium sp. L001.

[0072] (1) Preparation of polysaccharide polymer solution: Weigh a certain amount of biopolymer powder prepared in Example 3, dissolve it in an appropriate amount of water, place it in a 60°C water bath to swell completely, and prepare a L001 polysaccharide polymer solution with a mass concentration of 0.5%.

[0073] (2) Temperature resistance of L001 polysaccharide polymer: The viscosity of the polymer solution at 20℃, 40℃, 60℃, 80℃ and 100℃ was measured using an IKA rotational viscometer (rotor model: VOL-SP-6.7, rotation speed 5 rpm). As shown in Figure 8, the viscosity of the L001 polysaccharide polymer solution decreased with increasing temperature, especially when the temperature reached 80℃, the viscosity decreased significantly, indicating that L001 polysaccharide polymer is suitable for medium and low temperature oil reservoirs below 60℃.

[0074] (3) Salt resistance of L001 polysaccharide polymer: At room temperature, a certain amount of salt was added to the L001 polysaccharide polymer solution to achieve salinity of 2000, 5000, 10000, and 20000 mg / L. The viscosity at different salinity was measured using an IKA rotational viscometer (rotor model: VOL-SP-6.7, rotation speed 5 rpm). As shown in Figure 9, salt ions had little effect on the viscosity of the L001 polysaccharide polymer solution. In fact, under high salinity conditions, L001 polysaccharide exhibited a higher viscosity than under low salinity conditions, indicating that L001 polysaccharide is not significantly affected by salinity and is suitable for high-salinity oil reservoirs.

[0075] (4) pH tolerance range of L001 polysaccharide polymer: At room temperature, the viscosity of the polymer solution was measured at pH = 2, 4, 6, 8, 10, and 12 using an IKA rotational viscometer (rotor model: VOL-SP-6.7, rotation speed 5 rpm). As shown in Figure 10, the viscosity of the L001 polysaccharide polymer solution remained stable over a wide pH range (pH 2.0-10.0), exhibiting good stability, especially under acidic conditions, and retaining high viscosity even in a strongly acidic environment at pH 2.0. Example 9

[0076] This embodiment illustrates the response of L001 polysaccharide polymer solution to complex environmental factors such as high temperature, high salt, extreme pH, and crude oil.

[0077] (1) Preparation of biopolymer solution: Weigh a certain amount of L001 polysaccharide polymer powder prepared in Example 3, dissolve it in an appropriate amount of water, place it in a 60°C water bath to swell completely, and prepare a polysaccharide polymer solution with a mass concentration of 1.5%.

[0078] (2) Viscosity-temperature curves at different pH values ​​for a salinity of 5000 mg / L: At room temperature, a certain amount of salt was added to the biopolymer solution to achieve a salinity of 5000 mg / L. The pH values ​​of the solution were adjusted to 1, 4, 7, 10, and 12, respectively. The polymer solution was then kept at different temperatures (20℃, 40℃, 60℃, 80℃, and 100℃) for 24 hours. The viscosity of the polymer solution under these conditions was measured using an IKA rotational viscometer (rotor model: SP-3, rotation speed 30 rpm). As shown in Figure 11, under the same salinity, the viscosity retention rate was higher at pH values ​​of extremely acidic (pH1) and extremely alkaline (pH12) within the temperature range of 20-60℃. When the pH was 4-10, the viscosity decreased significantly with increasing temperature, indicating that this polysaccharide is suitable for extremely acidic and extremely alkaline reservoirs at low salinity.

[0079] (3) Viscosity-temperature curves at different pH values ​​with a salinity of 20,000 mg / L: At room temperature, a certain amount of salt was added to the biopolymer solution to achieve a salinity of 20,000 mg / L. The pH values ​​of the solution were adjusted to 1, 4, 7, 10, and 12, respectively. The polymer solution was kept at different temperatures (20℃, 40℃, 60℃, 80℃, and 100℃) for 24 hours. The viscosity of the polymer solution under these conditions was measured using an IKA rotational viscometer (rotor model: SP-3, rotation speed 30 rpm). As shown in Figure 12, with the continuous change of pH, the overall trend of the temperature-viscosity curve is very similar to that at a salinity of 5,000 mg / L, indicating that this polysaccharide is still suitable for extremely acidic and extremely alkaline reservoirs at high salinity.

[0080] (4) Viscosity-temperature curves at different pH values ​​with a mineralization of 50,000 mg / L: At room temperature, a certain amount of salt was added to the biopolymer solution to achieve a mineralization of 50,000 mg / L. The pH values ​​of the solution were adjusted to 1, 4, 7, 10, and 12, respectively. The polymer solution was then kept at different temperatures (20℃, 40℃, 60℃, 80℃, and 100℃) for 24 hours. The viscosity of the polymer solution under these conditions was measured using an IKA rotational viscometer (rotor model: SP-3, rotation speed 30 rpm). As shown in Figure 13, the temperature-viscosity curves show that, under the same mineralization, the viscosity retention rate is higher in the range of 20-60℃ when the pH is extremely acidic and extremely alkaline. This indicates that L001 polysaccharide has better viscosity retention characteristics in highly mineralized, extremely acidic, and extremely alkaline environments.

[0081] (5) Viscosity-temperature curves at different pH levels with a salinity of 50,000 mg / L and the presence of crude oil: At room temperature, a certain amount of salt was added to the biopolymer solution to achieve a salinity of 50,000 mg / L. Then, 1,000 ppm of crude oil was added, and the pH of the solution was adjusted to 1, 4, 7, 10, and 12, respectively. The polymer solution was kept at different temperatures (20℃, 40℃, 60℃, 80℃, and 100℃) for 24 hours. The viscosity of the polymer solution under these conditions was measured using an IKA rotational viscometer (rotor model: SP-3, rotation speed 30 rpm). As shown in Figure 14, the temperature-viscosity curves show that the viscosity of the biopolymer solution increased after the addition of crude oil, and it exhibited high viscosity retention rates over a wide range of pH levels, especially in acidic and extremely alkaline environments, demonstrating good application potential in harsh oil reservoirs. Example 10

[0082] This example illustrates the core simulation oil displacement effect of the L001 polysaccharide polymer solution.

[0083] The fermentation broth for producing extracellular polysaccharides from Rhizobium sp. L001 was obtained as described in Example 2. A certain amount of the L001 polysaccharide polymer fermentation broth was taken and diluted with tap water to a L001 polysaccharide content of 0.2% (w / v). A multifunctional steam and foam displacement experimental apparatus (ZQPM-II) was used, with an artificial loose core of 3.80 cm in diameter, 60.00 cm in length, and a porosity of 42.05%. Core conditions: saturated oil volume 275 mL; temperature: 60 °C; polymer flooding injection rate: 240 mL / h; displacement fluid injection rate: 240 mL / h. Methods: ① Blank group: After the model was saturated with oil, it was aged at 40 °C for 12 h, and then 10 PV of tap water was injected forward at 60 °C for displacement. ② Experimental group: After the model was saturated with oil, it was aged at 40 °C for 12 h, and then 6 PV of tap water was injected forward at 60 °C for displacement, followed by 4 PV of L001 polysaccharide polymer displacement.

[0084] Evaluation Results: During the oil displacement process, the oil displacement volume stopped increasing after 6 PV of water injection. At this point, L001 polysaccharide polymer was injected for oil displacement. Injection of 0.6 PV of L001 polysaccharide polymer resulted in the displacement of a significant amount of crude oil, with the improvement in oil displacement efficiency mainly concentrated within the range of 0.6-1.3 PV. The water displacement volume was 265 mL, with an oil displacement efficiency of 73.7%. The L001 polysaccharide polymer displacement volume was 274 mL, with an oil displacement efficiency of 79.8%. The oil displacement efficiency increased by 6.1%.

Claims

1. A rhizobium, classified as Rhizobium sp. L001, with accession number CCTCC NO: M20222005.

2. The application of the rhizobium of claim 1 in the production of polysaccharide biopolymers, characterized in that, The main components of the polysaccharide biopolymer are galactose and glucose, with a molar ratio of 1:

3.

3. The application according to claim 2, characterized in that, The polysaccharide biopolymer is used to prepare bioemulsifiers.

4. The application according to claim 2, characterized in that, The polysaccharide biopolymer is used for polymer flooding.

5. The application according to claim 4, characterized in that, The polysaccharide biopolymer is used in the form of fermentation broth for biopolymer flooding in oil reservoirs.

6. The application according to claim 5, characterized in that, The fermentation broth is obtained by fermenting rhizobia, and the fermentation broth is diluted to a preset mass concentration of polysaccharides before use.

7. The application according to claim 4, characterized in that, The polysaccharide biopolymer is used in reservoir environments with pH = 1-12.

8. The application according to claim 7, characterized in that, The polysaccharide biopolymer is used in extreme reservoir environments with pH=1-3 or pH=11-12.

9. The application according to claim 4, characterized in that, The polysaccharide biopolymer is used in oil reservoir environments at 20-60°C.

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