Preparation method of zinc oxide nanofluid with high salinity resistance based on halomonas-mediated synthesis and enhanced recovery application

Abstract

This invention provides a method for preparing a high-salinity zinc oxide-resistant nanofluid mediated by Halomonas sp. and its application in enhancing oil recovery. Specifically, it includes: a Halomonas sp. strain CDH-2, with accession number CCTCC NO: M 2025786, which can rapidly grow in high-concentration salt solutions and produce salt-resistant extracellular metabolites. Fermenting and culturing CDH-2 under high-salt conditions yields a fermentation broth with a surface tension below 30 mN / m. Mixing and culturing the obtained fermentation broth with a metal precursor solution yields a high-salinity-resistant metal nanofluid. The resulting metal nanofluid maintains good stable dispersibility in high-salinity water and effectively reduces oil-water interfacial tension. It alters the wettability of oil-wetting surfaces, emulsifies and disperses crude oil, and reduces crude oil viscosity. Therefore, it can be used as an enhanced oil recovery agent in high-salt reservoirs and is widely applied in oilfield flooding, well huff and puff, and heavy oil viscosity reduction.

Description

Technical Field

[0001] This invention belongs to the field of oilfield enhanced oil recovery, specifically involving a method for preparing a zinc oxide nanofluid mediated by Halomonas bacteria and its application in enhancing oil recovery. Background Technology

[0002] To address the challenges of residual oil development in oilfields, scholars both domestically and internationally have conducted extensive research on enhanced oil recovery (EOR) technologies and their field applications. Currently, nanofluid flooding agents are a research hotspot, with their oil displacement mechanisms primarily including reducing oil-water interfacial tension, improving rock wettability, and creating structural separation pressure. Due to their extremely small size, nanoparticles can easily penetrate microporous channels in low-permeability reservoirs, improving fluid migration efficiency and thus exhibiting unique advantages in adsorption flooding for enhanced oil recovery. However, the core bottleneck of nanofluid flooding agents lies in the tendency of nanoparticles to aggregate and precipitate, severely restricting the stability of the oil displacement system and directly affecting the effectiveness of nanofluid flooding.

[0003] Studies have shown that relying solely on traditional mechanical stirring cannot overcome the van der Waals interactions between nanoparticles, necessitating the exploration of a methodological system combining chemical modification strategies with physical field control methods. Among these, surface synthesis / modification techniques have demonstrated excellent control over the dispersion stability of nanoparticles. By leveraging the synergistic mechanism of special chemical bonds and steric hindrance, oriented shielding bands can be constructed while preserving the original activity of nanoparticles, effectively mitigating the problems of reverse adsorption and aggregation caused by opposing ions in solution. With the deep integration of nanotechnology and green chemistry, bio-based green synthesis of nanoparticles has become a core research direction for addressing environmental pressures and optimizing nanoparticle synthesis / modification processes. These methods utilize bioactive substances or metabolites secreted by organisms as reducing agents and stabilizers, synthesizing nanoparticles through bio-mediated synthesis. This endows nanoparticles with unique functionalized surface properties, resulting in good stable dispersion. However, bioactive substances are prone to structural degradation under extreme environments, leading to changes in the functionalized surface properties of nanoparticles and consequently, a decrease in their stability and functionality in fluids. As is well known, most oilfield formations are high-salinity / high-mineralization environments. During the oil displacement process, nanofluids inevitably encounter these extreme high-mineralization environments. The high concentration of ions in the oil displacement agent fluid compresses the diffusion double layer of nanoparticles, leading to instability and aggregation, which significantly affects the oil displacement effect of the nano-displacement agent. Therefore, using bioactive substances produced by salt-tolerant microorganisms for green synthesis, endowing nanofluids with high stability in high-salinity environments, has broad application prospects in the field of oilfield nano-displacement agents.

[0004] In summary, there is an urgent need to find a strain with strong salt tolerance and whose metabolites can be used to synthesize nanoparticles, in order to enhance the stability of nanofluids under high salinity reservoir conditions, and thus expand the application of nanofluids in the field of improving reservoir recovery. Summary of the Invention

[0005] To address the aforementioned issues, this invention screened a Halomonas sp. CDH-2 strain that can grow under high-salt conditions and whose metabolites can be used to synthesize nanoparticles. It can grow in 20-200 g / L sodium chloride medium, and its growth metabolites are glycolipid surfactants. Its fermentation broth can be used to mediate the synthesis of nanofluids resistant to high salinity and to improve the recovery rate of salt / high salinity oil reservoirs.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention provides a Halomonas sp. strain CDH-2, which was deposited on April 15, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025786.

[0008] Preferably, the nucleotide sequence of the 16S rRNA of the *Halomonas* strain CDH-2 is 1535 bp, as shown in SEQ ID NO. 1.

[0009] Preferably, the Halomonas CDH-2 has the following characteristics:

[0010] (1) Morphological characteristics: Gram-negative bacteria, non-spore-forming, rod-shaped, without flagella, smooth surface, individual length between 0.8-2.5 μm;

[0011] (2) Colony characteristics: The colonies are light pink, round in shape with very irregular edges, and moist with wrinkles on the surface;

[0012] (3) Growth characteristics: aerobic, chemoheterotrophic, growth temperature is 4-40 ℃;

[0013] (4) Salt tolerance: It can grow in a medium with a NaCl concentration of 20-200 g / L.

[0014] Preferably, the preferred growth temperature of the halometabolite CDH-2 is 35-38 ℃ and the pH tolerance value is 6.9-7.5; more preferably, the growth temperature of the halometabolite CDH-2 is 37 ℃ and the pH tolerance value is 7.0.

[0015] The present invention provides a fermentation broth of the aforementioned Halomonas CDH-2.

[0016] The present invention provides a method for preparing the fermentation broth of *Haloxymonas salina* CDH-2, specifically comprising: inoculating *Haloxymonas salina* CDH-2 into a high-salt culture medium, carrying out fermentation culture, and collecting the fermentation broth to obtain the product.

[0017] Preferably, the high-salt culture medium comprises the following components: NaCl 20-200 g / L, glucose 25-35 g / L, NaNO3 8-12 g / L, Na3C6H5O7 2-4%, KH2PO4 0.5-1.5 g / L, (NH4)2HPO4 0.5-1.5 g / L, MgSO4·7H2O 0.1-0.3 g / L, and Na2MoO4 0.02-0.14 g / L; more preferably, the pH of the high-salt culture medium is 7.0-7.5.

[0018] Most preferably, the high-salt culture medium comprises the following components: NaCl 100 g / L, glucose 30 g / L, NaNO3 10 g / L, Na3C6H5O7 3%, KH2PO4 1 g / L, (NH4)2HPO4 1 g / L, MgSO4·7H2O 0.2 g / L, Na2MoO4 0.08 g / L, with a pH of 7.0-7.2.

[0019] Preferably, the fermentation temperature is 35-40 ℃ and the fermentation time is 1-7 days; more preferably, the fermentation temperature is 36-37 ℃ and the fermentation time is 3-5 days; most preferably, the fermentation temperature is 37 ℃ and the fermentation time is 3 days.

[0020] Preferably, the fermentation culture conditions are shaker culture; preferably, the rotation speed of the shaker is 150-300 rpm, more preferably, the rotation speed of the shaker is 180 rpm.

[0021] Preferably, the OD of the fermentation broth of the *Haloxylon ammodendron* CDH-2 is... 600 The value is 0.8-1.0.

[0022] Preferably, the surface tension of the fermentation broth of the halomonas CDH-2 is below 30 mN / m.

[0023] This invention provides a method for synthesizing highly mineralizable metal nanofluids based on the *Haloxylon ammodendron*-mediated synthesis, specifically comprising: mixing and culturing the *Haloxylon ammodendron* CDH-2 fermentation broth with a metal precursor solution to obtain highly mineralizable nanofluids.

[0024] Preferably, the CDH-2 halophilus fermentation broth needs to be filtered before use to remove bacterial interference. Specifically, conventional methods known to those skilled in the art can be used, such as using a 0.22 μm vacuum filter membrane to filter the fermentation broth and collecting the filtrate to remove bacterial interference.

[0025] Preferably, the molar concentration of metal ions in the metal precursor solution is 20-40 mM, and the pH is 7.0-7.2; more preferably, the molar concentration of metal ions in the metal precursor solution is 30 mM; even more preferably, the metal ions are zinc ions. This invention does not specifically limit the source of zinc ions; conventional zinc ion reagents in the art, such as zinc acetate or zinc nitrate, can be used. This invention does not specifically limit the preparation of the zinc ion solution; conventional solution preparation methods in the art can be used, adjusting the pH of the precursor solution to 7.0-7.2 with NaOH. The specific molar concentration of metal ions in the aforementioned metal precursor solution designed in this invention is beneficial for obtaining metal nanoparticles with smaller particle sizes.

[0026] Preferably, the volume ratio of the metal precursor solution to the fermentation broth is 1:(1-1.5); more preferably, the volume ratio of the metal precursor solution to the fermentation broth is 1:1.25.

[0027] Preferably, the mixed culture temperature is 35-40 ℃; the mixed culture time is 12-48 h; more preferably, the mixed culture temperature is 37 ℃; the mixed culture time is 36 h.

[0028] Preferably, the culture is carried out in a shaker; more preferably, the rotation speed of the shaker is 150-300 rpm; most preferably, the rotation speed of the shaker is 180 rpm.

[0029] The present invention provides a high-mineralization-resistant metal nanofluid synthesized by the above method. Preferably, the metal nanoparticles in the metal nanofluid have a particle size of 30-60 nm. More preferably, the high-mineralization-resistant metal nanofluid is a zinc oxide nanofluid.

[0030] This invention provides a high-mineralization-resistant metal nanofluid synthesized via the above-described preparation method. Specifically, the active substances containing amino and carboxyl functional groups in the CDH-2 strain fermentation broth can react with metal ions (such as Zn). 2+ The ligands act as ligands in the synthesis of ZnO NPs, influencing their formation, size, and morphology. Meanwhile, a large amount of organic polymer polysaccharides can prevent the aggregation of metal nanoparticles, forming a stable diffused double layer structure. This allows the synthesized metal nanoparticles to be uniformly and stably dispersed in the fermentation broth of strain CDH-2, forming a stable solid-liquid colloidal solution.

[0031] Meanwhile, the high-saltability resistant metal nanofluid synthesized in this invention maintains good stability in high-saltability water. The main reason for this is that the active components in the fermentation broth of the CDH-2 strain are tolerant to high-salt environments. Due to the unique salt-tolerant genes and mechanisms of the CDH-2 strain, the active components metabolized in high-salt environments possess excellent salt-tolerant properties. These salt-tolerant active substances participate in the synthesis process of metal nanoparticles, resulting in the synthesized nanoparticles being grafted / encapsulated with salt-tolerant active substances. The diffusion double-layer structure of the nanoparticles is not significantly affected by salinity in high-salt environments, resulting in a more stable structure and thus achieving stable dispersion of the nanoparticles under high-salt conditions.

[0032] In summary, the method for preparing high-mineralization-resistant metal nanofluids mediated by Halomonas provided by this invention has many advantages, such as simple and mild preparation process, low cost, and stable properties of the obtained metal nanoparticles in high-mineralization oil reservoir formation water.

[0033] This invention also provides the application of the above-mentioned high-salinity resistant metal nanofluid in enhancing oil recovery in high-salinity reservoirs. Specifically, the high-salinity resistant metal nanofluid synthesized by this invention can significantly reduce the oil-water interfacial tension under high-salinity conditions and has functions such as changing reservoir wettability, emulsifying and stripping crude oil. Therefore, it can be used as an enhanced oil recovery agent and is widely applied in oilfield flooding, well huff and puff, and heavy oil viscosity reduction.

[0034] Preferably, when the high-mineralization-resistant metal nanofluid is used in an oil displacement process, the parameters include: injecting the high-mineralization-resistant metal nanofluid at an injection rate of 10-30 μL / min into an area with a permeability of 5-10 × 10⁻⁶ μL / min. -3 μm 2 In water-wet reservoirs, the injection volume is 0.4-0.6 PV of the reservoir pore volume. The injection sequence is as follows: after a first water flood to a water cut of 90%, the nanofluid is injected, followed by a second water flood. After displacement by the high-mineralization-resistant metal nanofluid, the recovery rate increases by more than 40% compared to a single water flood.

[0035] Preferably, when the high-mineralization-resistant metal nanofluid is used for oil well huff and puff, the parameters include: injecting the high-mineralization-resistant metal nanofluid into the oil reservoir at an injection volume of 0.1-0.5 times the pore volume of the oil layer, and then simmering the well at 60-90 ℃ for 24-72 h after injection, and then opening the well to produce oil after the simmering period.

[0036] Preferably, when the high-mineralization-resistant metal nanofluid is used to reduce the viscosity of heavy oil, the parameters include: mixing the high-mineralization-resistant metal nanofluid with crude oil in the wellbore at a mass ratio of (1-5):1, and contacting it at 35-45 ℃ and 100-200 r / min for 24-96 hours; after treatment, the viscosity of crude oil at 40 ℃ is reduced from 2264.34 mPa·s and 12900.17 mPa·s to 56.23 mPa·s and 89.12 mPa·s, respectively, with a viscosity reduction rate >97%.

[0037] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0038] This invention provides a Halomonas sp. strain CDH-2, with accession number CCTCCNO: M 2025786. Fermentation culture of the Halomonas sp. strain CDH-2 provided by this invention in a high-concentration salt solution yields a fermentation broth with a surface tension below 30 mN / m. The surfactants and emulsifiers contained in the fermentation broth can act as reducing agents and stabilizers for nanoparticles, enabling the preparation of nanoparticles and / or metal oxide nanoparticles, achieving green-mediated synthesis of nanoparticles. The synthesized nanoparticles are uniformly dispersed in fluids and maintain stable dispersion even in highly salinized formation water.

[0039] In summary, the Halomonas sp. strain CDH-2 provided by this invention can be specifically used to prepare nanoparticles resistant to high mineralization. The Halomonas sp. strain CDH-2 provided by this invention has broad application prospects in the green-mediated synthesis of high-mineralization-resistant nanoparticles from petroleum and natural gas.

[0040] Furthermore, the fermentation broth of the Halomonas sp. strain CDH-2 provided by this invention is mixed with zinc ions and cultured. The surfactant and emulsifier components in the fermentation broth, together with the nanoparticles synthesized under their mediated conditions, form a high-salinity resistant nanofluid system. This system can significantly reduce the oil-water interfacial tension under high-salinity conditions and change reservoir wettability. The high-salinity resistant nanofluid system can be used as a high-salinity resistant nanofluid extraction agent, and can be applied to oilfield flooding, huff and puff, and heavy oil viscosity reduction. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 Photograph of LB plate colonies of Halomonas CDH-2 strain;

[0043] Figure 2 Electron micrograph of Halomonas CDH-2 strain;

[0044] Figure 3 Phylogenetic tree of 16S rRNA in the *Halomonas* CDH-2 strain;

[0045] Figure 4 FTIR image of fermentation broth of Halomonas CDH-2 strain;

[0046] Figure 5 Figure 1. Particle size distribution and morphology of metal oxide nanoparticles in a zinc oxide nanofluid with high mineralization resistance.

[0047] Figure 6 EDS diagram of metal oxide nanoparticles in zinc oxide nanofluid with high mineralization resistance;

[0048] Figure 7 Figure showing the variation of wettability at the interface between high-mineralization zinc oxide nanofluid and formation water-oil wetting fluid.

[0049] Figure 8 Macroscopic and quantitative analysis of the oil displacement effect of highly mineralized zinc oxide nanofluids;

[0050] Figure 9 The image shows the emulsification effect of highly mineralized zinc oxide nanofluids on chain hydrocarbon substrates.

[0051] Figure 10 Microscopic morphology diagram of the viscosity reduction effect of highly mineralized zinc oxide nanofluid on crude oil. Detailed Implementation

[0052] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0053] Unless otherwise specified, the technical means used in this invention are conventional means well known to those skilled in the art. All raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. Unless otherwise specified, all reagents used in this invention are of analytical grade.

[0054] Example 1

[0055] 1. Screening of Halomonas sp. CDH-2

[0056] Oil and water samples were collected from the oilfield. 10 g of the collected liquid was added to 200 mL of LB medium with a NaCl concentration of 100 g / L. The mixture was incubated at 37 ℃ with shaking at 150 rpm for 2 days. After 2 days, 100 μL of the supernatant was evenly spread onto LB agar plates containing a NaCl concentration of 100 g / L. After incubation at 37 ℃ for 2 days, single colonies were selected and purified 3-4 times on LB agar plates. The purified colonies were then mixed with LB liquid medium containing 30% glycerol and stored at -80 ℃. The strain was identified as *Halomonas* sp. by 16S rRNA sequencing and named CDH-2. Further identification and analysis were then performed.

[0057] 2. Identification of Halomonas sp. CDH-2

[0058] 2.1 Morphological observation of Halomonas sp. CDH-2

[0059] Strains CDH-2 were cultured on solid LB medium at 37 ℃ for 72 h, and the colony morphology of the strain was observed as follows. Figure 1 As shown; scanning electron microscopy was performed on the bacterial cells, and the results are as follows. Figure 2 As shown.

[0060] Depend on Figure 1 and Figure 2 It was found that strain CDH-2, after growing in solid LB medium for 72 h, produced circular colonies with extremely irregular edges, a moist and wrinkled surface, and a light pink color. Scanning electron microscopy revealed that the bacterial cells were rod-shaped, with short rod-shaped spores, no flagella, a smooth surface, and individual lengths ranging from 0.8 to 2.5 μm.

[0061] Further culturing of the strain determined that the optimal temperature range for the growth of strain CDH-2 was 35-38℃, and the pH tolerance value was 6.9-7.5.

[0062] 2.2 Molecular identification

[0063] The 16S rRNA gene fragment of strain CDH-2 was amplified by colony PCR, and its DNA sequence was obtained by Sanger sequencing. The 16S rDNA sequence of strain CDH-2 is shown in SEQ ID NO.1, specifically:

[0064]

[0065] Comparison analysis of 16S rRNA nucleotide sequences with the NCBI database is performed; generally, a sequence similarity of 97% or higher is considered to indicate the same bacterial species. A phylogenetic tree based on the 16S rRNA gene sequence is shown below. Figure 3 As shown. Figure 3 The Halomonas sp. CDH-2 in the sample showed a 98% similarity to Halomonas desiderata strain D167-6-1, thus identifying strain CDH-2 as Halomonas. Therefore, based on morphological characteristics and molecular biological 16S rRNA gene sequencing results, strain CDH-2 was confirmed to be Halomonas.

[0066] Example 2

[0067] The preparation of the fermentation broth of Halomonas sp. CDH-2 is as follows:

[0068] Halomonas sp. CDH-2 was inoculated onto a fermentation medium and cultured on a shaker. The fermentation broth was then collected to obtain the product.

[0069] The fermentation medium used consisted of the following raw materials: NaCl 100 g / L, glucose 30 g / L, NaNO3 10 g / L, Na3C6H5O7 3%, KH2PO4 1 g / L, (NH4)2HPO4 1 g / L, MgSO4·7H2O 0.2 g / L, Na2MoO4 0.08 g / L, pH 7.0, with the remainder being deionized water.

[0070] The temperature for shaking culture was 37℃, the shaking culture time was 3 days, and the shaking speed was 180 rpm.

[0071] After the shaker culture was completed, the fermentation broth of Halomonas sp. CDH-2 was obtained, with an OD600nm of 0.91.

[0072] Example 3

[0073] The surface tension of the fermentation broth of Halomonas sp. CDH-2 was tested, as follows:

[0074] Zero the surface tension meter according to the instructions, using deionized water (72 mN / m) as a blank control. Pipette 30 mL of the Halomonas sp. CDH-2 fermentation broth prepared in Example 2 into the surface tension meter's measuring chamber. The liquid pulls the platinum plate downwards until the test value stabilizes. Record the surface tension of the sample as 29.6 mN / m.

[0075] Compared with the surface tension of deionized water (72 mN / m), the surface tension of CDH-2 fermentation broth was significantly lower, indicating that the fermentation broth of Halomonas sp. CDH-2 may contain surfactant components.

[0076] Example 4

[0077] The metabolites of the fermentation broth of Halomonas sp. CDH-2 were analyzed as follows:

[0078] The fermentation broth of *Halomonas* sp. CDH-2 prepared in Example 2 was filtered through a 0.22 μm filter to remove bacterial interference. The filtrate was then freeze-dried under vacuum (5 MPa, -20°C) for 48 h to obtain a powder. Using the potassium bromide pelleting method, 1.5273 mg of the powder sample and 200 mg of pure KBr were collected, uniformly ground, placed in a mold, and pressed into transparent sheets using a hydraulic press. The sample was then tested using a Thermo Scientific Nicolet 6700 FTIR spectrometer at wavelengths between 4000 and 400 cm⁻¹. -1 The spectrum was measured within the specified range, with 32 scans and a resolution of 4 cm⁻¹. -1 .

[0079] The FTIR spectrum of the Halomonas sp. CDH-2 fermentation broth prepared in Example 2 is shown below. Figure 4 As shown in the figure, the infrared spectrum of the Halomonas sp. CDH-2 fermentation broth is as follows: 3776.59, 3695.69, 3423.73, 2923.62, 1645.12, 1439.59, 1385.39, 1143.59, 994.04 cm⁻¹ -1 Characteristic peaks are observed at 3776.59 and 3695.69 cm⁻¹. -1 The absorption peak at 3423.73 cm⁻¹ is due to the stretching vibration of free OH groups, indicating the presence of a special hydrogen bond structure. -1The broad absorption peak at 2923.62 cm⁻¹ corresponds to the stretching vibration of intermolecular hydrogen bonds (OH bonds), indicating the presence of alcohol or phenolic groups, corresponding to the hydroxyl groups in glycolipid moieties or carboxylic acid groups in fatty acids. Alkenes, alkynes, and aromatic compounds may also be present. -1 The absorption peak at 1645.12 cm⁻¹ reflects the stretching vibration of CH, especially the -CH₂- / -CH₃- groups in saturated fatty acids, indicating the presence of alkane compounds, commonly found in the hydrophobic tails of fatty acids or glycolipids. -1 The absorption peaks at these locations are typically associated with C=C stretching vibrations, indicating the presence of compounds such as olefins or aromatic rings, which may be unsaturated fatty acids in glycolipids. (1439.59 and 1385.39 cm⁻¹) -1 The absorption peak at 1143.59 cm⁻¹ exhibits the bending vibration characteristics of CH, particularly the -CH₂- / -CH₃- bending vibration mode, indicating the presence of alkanes and alkenes, commonly found in fatty acid biosurfactants. -1 The absorption peak at 994.04 cm⁻¹ corresponds to the stretching vibration of CO, which is related to ester bonds or ether bonds in sugar rings. It is commonly seen in the CO vibration of sugar groups in glycolipids (such as glycosidic bonds) and is a characteristic structure of glycolipids. -1 The absorption peak at this point is related to the COC vibration or the bending vibration of olefins (CH2=CH2), indicating the presence of olefinic compounds, which are commonly found in the characteristic vibrations of sugar rings in glycolipids (such as the COC of rhamnose). Based on the analysis of the above characteristic peaks in relevant literature, the biosurfactant produced by the fermentation metabolism of Halomonas sp. CDH-2 is a glycolipid surfactant, characterized by a hydrophilic glycosyl group and a hydrophobic fatty chain, possibly containing unsaturated structures and ester bonds.

[0080] In summary, the presence of these active functional groups enables the fermentation broth of Halomonas sp. CDH-2 to effectively prevent nanoparticle aggregation and promote the stability of nanoparticles in the culture medium during the process of mediating nanoparticle synthesis.

[0081] Example 5

[0082] A method for preparing zinc oxide nanofluids with high mineralization resistance mediated by Halomonas is as follows:

[0083] Preparation of metal precursor solution: Adjust the pH of the precursor solution to 7 with NaOH to obtain a 30 mM zinc acetate hexahydrate solution (if necessary, zinc acetate hexahydrate can be added to maintain the zinc ion concentration).

[0084] Preparation of fermentation broth of Halomonas sp. strain CDH-2 with cell removal: The fermentation broth of Halomonas sp. strain CDH-2 prepared in Example 2 was filtered using a 0.22 μm vacuum filter membrane, and the filtrate was collected to remove cell interference.

[0085] Under magnetic stirring conditions, the fermentation broth with the bacterial cells removed was mixed with the metal precursor solution at a mass ratio of 1.25:1. The mixture was then placed in a constant temperature shaker at 37 ℃ and 180 rpm for 36 h to obtain the high-mineralization zinc oxide nanofluid oil displacement agent mediated by Halomonas.

[0086] Example 6

[0087] The particle size of the metal oxide nanoparticles in the high-mineralization-resistant zinc oxide nanofluid prepared in Example 5 was measured using a laser particle size analyzer, as detailed below:

[0088] Pour the high-mineralization-resistant zinc oxide nanofluid prepared in Example 5 into a clean cuvette. The liquid level should be about 2 / 3 of the height of the cuvette. Note that the liquid level must cover the laser beam 5 mm above the cuvette.

[0089] Adjust the laser particle size analyzer according to the instrument's instruction manual and run the main program.

[0090] The particle size distribution of metal oxide nanoparticles in the high-mineralization zinc oxide nanofluid was detected as follows: Figure 5 As shown in the left figure, the particle size of the zinc oxide nanofluid prepared in Example 5 ranges from 30 to 60 nm, with an average particle size of 41.3 nm.

[0091] Example 7

[0092] The size, distribution, and morphology of the nanoparticles were characterized using scanning electron microscopy (SEM), as detailed below:

[0093] Take 5 mL of the zinc oxide nanofluid with high mineralization resistance prepared in Example 5 into a centrifuge tube, centrifuge (10000 rpm, 10 minutes), discard the supernatant, then add 1 mL of anhydrous ethanol to the centrifuge tube, sonicate at room temperature for 5 minutes, take a small amount of liquid drop onto a silicon wafer, dry it, and then test it.

[0094] Adjust the scanning electron microscope according to the instrument's instruction manual, and perform the following operations in sequence: venting, vacuuming, voltage increase, and sample stage raising. After positioning the sample, scan the image according to the magnification and image scale and save it.

[0095] The particle size and morphology of metal oxide nanoparticles in the high-mineralization zinc oxide nanofluid were detected as follows: Figure 5 As shown in the right figure, under a scanning electron microscope at 100,000× magnification, the surface of the zinc oxide nanofluid prepared in Example 5 exhibits a relatively rough and porous structure, with a spherical shape. Nanoparticles of different sizes aggregate and accumulate due to the interaction forces between surface functional groups.

[0096] Example 8

[0097] The elemental composition of the metal oxide nanoparticles in the high-mineralization-resistant zinc oxide nanofluid prepared in Example 5 was determined using X-ray energy dispersive spectroscopy, as follows:

[0098] Case 5: Preparation of High-Mineralization-Resistant Zinc Oxide Nanofluid via Vacuum Freeze-Drying, yielding a powdered material. A very thin layer of gold was sprayed onto the surface of the powdered sample to ensure good conductivity. The elemental composition of the metal oxide nanoparticles was determined according to the instructions of the EDX detector connected to the SEM instrument (Oxford Instruments), thus confirming the material composition of the metal oxide nanoparticles. The detection results are as follows... Figure 6 As shown in Table 1.

[0099] Table 1. Elemental composition of metal oxide nanoparticles in the high-mineralization-resistant zinc oxide nanofluid prepared in Example 5

[0100]

[0101] Depend on Figure 6 As can be seen, the elemental composition of the zinc oxide nanoparticles in the *Halomonas* sp.-mediated synthesis of highly mineralized zinc oxide nanofluid prepared in Example 5 is: C (52.4%), O (23.4%), and Zn (24.2%). The C element mainly consists of residual carbon-containing culture medium from the *Halomonas* sp. strain CDH-2 fermentation broth and metabolic products of *Halomonas* sp. strain CDH-2 fermentation (such as polysaccharides, proteins, and lipids). During the synthesis of zinc oxide nanoparticles, these carbon-containing organic compounds are adsorbed / coated or participate in zinc oxide synthesis, thereby encapsulating / grafting onto the surface of the nanoparticles, resulting in a high carbon content in the elemental composition of the nanoparticles. Therefore, the active substances in the *Halomonas* sp. strain CDH-2 fermentation broth have the potential to synthesize metal oxide nanoparticles.

[0102] Examples 9-16

[0103] A method for testing the mineralization resistance of a halophilic nanofluid synthesized with high mineralization resistance to zinc oxide is described below:

[0104] 1. Prepare simulated mineralized water with mineralization values ​​of 20,000 mg / L, 80,000 mg / L, 130,000 mg / L, and 200,000 mg / L:

[0105] a. 20000 mg / L mineralized water: Weigh 1.73 g calcium chloride (CaCl2), 1.17 g magnesium chloride (MgCl2), 5.27 g sodium sulfate (Na2SO4), and 11.83 g sodium chloride (NaCl) into beakers, and dissolve and dilute them with distilled water to a total volume of 1 L (all reagents used are chemically pure).

[0106] b. 80000 mg / L mineralized water: Weigh 6.93 g calcium chloride (CaCl2), 4.67 g magnesium chloride (MgCl2), 21.07 g sodium sulfate (Na2SO4), and 47.33 g sodium chloride (NaCl) into beakers, and dissolve and dilute them with distilled water to a total volume of 1 L (all reagents used are chemically pure).

[0107] c. 130000 mg / L mineralized water: Weigh 11.27 g calcium chloride (CaCl2), 7.58 g magnesium chloride (MgCl2), 34.24 g sodium sulfate (Na2SO4), and 76.91 g sodium chloride (NaCl) into beakers, and dissolve and dilute them with distilled water to a total volume of 1L (all reagents used are chemically pure).

[0108] d. 200,000 mg / L mineralized water: Weigh 17.3 g calcium chloride (CaCl2), 11.7 g magnesium chloride (MgCl2), 52.7 g sodium sulfate (Na2SO4), and 118.3 g sodium chloride (NaCl) into beakers, and dissolve and dilute them with distilled water to a total volume of 1 L (all reagents used are chemically pure).

[0109] 2. Collect formation water from oilfields with different mineralization: Collect formation water with different mineralization from oilfields such as Changqing, Jianghan, Liaohe, Tuha, and Shengli.

[0110] Table 2. Mineralization, pH, and ionic composition of formation water collected from different oilfields.

[0111]

[0112] The formation water from the oilfields with different salinity was centrifuged (10,000 rpm, 10 min) to remove suspended impurities.

[0113] 3. Under magnetic stirring conditions, the high-mineralization-resistant zinc oxide nanofluid synthesized by Halomonas prepared in Example 5 was mixed with water of different mineralization and oilfield formation water at a volume ratio of 1:1, which was recorded as the mixture of Examples 9-16. Then the mixture was left to stand for different times.

[0114] 4. The particle size of the metal oxide nanoparticles after mixing the high-mineralization-resistant zinc oxide nanofluids prepared in Examples 9-16 with water of different mineralization and oilfield formation water was measured using a laser particle size analyzer. Specifically, the mixtures prepared in Examples 9-16 were poured into a clean cuvette, with the liquid level approximately 2 / 3 of the cuvette's height. It was ensured that the liquid level covered the laser beam position 5 mm above the cuvette. The laser particle size analyzer was adjusted according to the instrument's instruction manual, and the main program was run. The particle sizes of the metal oxide nanoparticles in the mixtures prepared in Examples 9-16 are shown in Table 3.

[0115] Table 3. Particle size of nanoparticles in mixtures of simulated water with different mineralization, oilfield formation water and zinc oxide nanofluid with high mineralization.

[0116]

[0117] Table 3 shows that the high-salinity zinc oxide nanofluid synthesized by the fermentation broth of *Halomonas* sp. strain CDH-2, after being mixed with simulated formation water of different salinities and left for 7 days, showed no precipitation in the colloidal solution. Although the nanoparticle size increased slightly with increasing salinity, the increase was small. After mixing with simulated formation water with a salinity of 20,000 mg / L, the nanoparticle size only increased from 48.72 nm to 51.25 nm. After mixing with formation water from the Jianghan Oilfield with a salinity of 249,429 mg / L and left for 7 days, the nanoparticle size was 55.34 nm.

[0118] The results showed that the nanoparticles in the high-salinity-tolerant zinc oxide nanofluid oil displacement agent synthesized by *Haloxymonas* exhibited good dispersion stability in high-salinity water, and the high concentration of mineral ions in the solution had little effect on the diffuse double-layer structure of the nanoparticles. This indicates that during the *Haloxymonas*-mediated synthesis of zinc oxide nanofluid, biomolecules generated by *Haloxymonas* metabolism participate in the synthesis process, resulting in the grafting / coating of these bioactive molecules insensitive to high salinity onto the synthesized zinc oxide nanoparticles, thus enabling the synthesized zinc oxide nanofluid to tolerate high salinity.

[0119] Example 17

[0120] The ability of the CDH-2 fermentation broth prepared in Example 2 and the *Halomonas* strain prepared in Example 5 to reduce oil-water interfacial tension was evaluated using an interfacial tensiometer based on the spin-drop method. The specific results are as follows:

[0121] CDH-2 fermentation broth and high-mineralization zinc oxide nanofluid were mixed with formation water from Jianghan Oilfield at a volume ratio of 1:1 to form the experimental group, while the formation water from Jianghan Oilfield served as the control group. Samples were prepared and the instrument was operated according to the instructions for use of the interfacial tensiometer. The length and width of oil droplets were measured every 10 minutes, and the interfacial tension was calculated. The measurements were repeated three times, and the average value and the interfacial tension between the nano-displacement agent and crude oil at different concentrations were taken. The results are shown in Table 4.

[0122] Table 4. Results of oil-water interfacial tension in formation water, fermentation broth of Halomonas sp. strain CDH-2, and the high-mineralization-resistant zinc oxide nanofluid prepared in Example 5.

[0123]

[0124] Table 4 shows that the oil-water interfacial tension of the high-salinity zinc oxide nanofluid synthesized by *Haloxylon ammodendron* is only 0.0759 mN / m, which is significantly lower than the oil-water interfacial tension of 0.8345 mN / m in oilfield formation water. This indicates that the *Haloxylon ammodendron*-mediated synthesis of the high-salinity zinc oxide nanofluid of this invention can effectively reduce the oil-water interfacial tension and demonstrates oil displacement potential.

[0125] Example 18

[0126] The ability of the high-mineralization zinc oxide nanofluid prepared in Example 5 to alter the wettability of the oil-wetting interface was determined using a contact angle meter.

[0127] The liquid phases were simulated formation water with a salinity of 20476 mg / L and the high-salinity zinc oxide nanofluid prepared in Example 5, respectively; the oil phase was simulated crude oil. The specific experimental steps are as follows:

[0128] Quartz glass slides were pretreated by immersing them in crude oil and then aged in an oven at 60 ℃ for 12 days before being taken out for use.

[0129] Clean the glass slide with n-heptane in a fume hood until the surface is slightly yellow or colorless, then wait for the n-heptane on the glass slide surface to evaporate, resulting in an oil-wetted glass slide;

[0130] Before the experiment, the oil-wetting interface glass slides were immersed in the high-mineralization-resistant zinc oxide nanofluid oil displacement agent prepared in Example 5 for 1 day, 3 days, 5 days and 7 days to evaluate the effect of the synthesized high-mineralization-resistant zinc oxide nanofluid on the wettability of the glass slides under different immersion time conditions; at the same time, the oil-wetting glass slides with the same pretreatment were immersed in simulated formation water for 0 days and 7 days as wettability control samples.

[0131] To study the degree of contact angle change using the pendant drop method, the oil-wetted glass slides that had been soaked and treated with different immersions were placed in the sample tank of the contact angle measuring instrument, and the zinc oxide nanofluid with high mineralization resistance prepared in Example 5 was added to submerge the glass slides.

[0132] Start the image measurement and analysis equipment, and adjust the position of the sample slot according to the image displayed on the screen to ensure that the experimental sample can be clearly displayed and recorded on the monitor.

[0133] Simulated crude oil (5 μL) was slowly added dropwise to the lower surface of a glass slide in the sample cell using a bent needle. After the oil droplets stabilized, a photograph was taken to record the contact angle value, which was then obtained from the software.

[0134] The results of the changes in wettability at the simulated formation water wetting interface of the high-mineralization zinc oxide nanofluid obtained in Example 18 are shown in Table 5 and... Figure 7 As shown.

[0135] Table 5. Contact angle data of high-mineralization zinc oxide nanofluid and simulated formation water in Example 15.

[0136]

[0137] Table 5 shows that the contact angle of the oil-wetted glass slide with formation water (0 days) was 144.2°, confirming that the initial state of the glass surface was oil-wetted. After immersion in formation water for 7 days, the oil-water-solid contact angle of the oil-wetted glass interface was 120°, which served as a control group. The contact angles of the high-mineralization zinc oxide nanofluid after immersion for 1, 3, 5, and 7 days were 70.0°, 52.2°, 38.8°, and 36.9°, respectively. The contact angle test results indicate that the high-mineralization zinc oxide nanofluid prepared in this invention has a significant wetting reversal effect on the oil-wetted interface. The reason for this is that the nanoparticles in the high-mineralization zinc oxide nanofluid have a large specific surface area and high surface activity, which allows them to be more firmly adsorbed onto the surface of the oil-wetted glass slide, significantly reducing the oleophilicity of the surface and enhancing its hydrophilicity. This stable nanoparticle adsorption layer can effectively change the wettability of the core surface, helping to reduce oil film adhesion and improve oil displacement efficiency.

[0138] Example 19

[0139] Microscopic displacement experiments were conducted using a micro-etched glass model to evaluate the oil displacement capability of the high-mineralization zinc oxide nanofluid synthesized in Example 5.

[0140] The oil phase was simulated as oil, and the water phase was simulated as formation water. The micro-etched glass model had dimensions of 68×68×4 mm, a depth of 30 μm, a pore diameter of 100-200 μm, and a permeability of 5-10×10⁻⁶. -3μm 2 All components exhibit water-wetness. The micromodel displacement steps are as follows:

[0141] The micro-etched glass model was cleaned multiple times with dichloromethane until all impurities in the pore throat channels were completely removed. Then, it was rinsed multiple times with deionized water to ensure the cleanliness of the micro-etched glass model.

[0142] The cleaned micro-etched glass model was placed in an oven at 80 ℃ for drying to ensure that the organic solvents completely evaporated.

[0143] Simulated oil was injected into the micromodel at a constant flow rate of 20 μL / min using a micro-pump, ensuring full contact between the oil and the inner wall of the micro-etched glass model until oil flowed continuously from the outlet. Subsequently, the micro-etched glass model was aged at 60 °C for 12 hours.

[0144] After the aging process, simulated formation water was injected from the inlet of the micro-etched glass model at the same flow rate for one waterflooding operation until water continued to flow from the outlet. The remaining oil morphology of the overall micro-etched glass model and local areas after waterflooding was photographed and recorded using a camera and microscope.

[0145] Next, 0.5 PV of high-mineralization zinc oxide nanofluid was injected at the same flow rate.

[0146] Ensure sufficient contact between the high-salinity zinc oxide nanofluid and the simulated oil and pore throat channels. Record the state of the remaining oil during the high-salinity zinc oxide nanofluid displacement process by taking photographs.

[0147] Finally, simulated formation water was injected at the same flow rate for a second waterflooding operation until no more oil was produced at the outlet. The displacement was then stopped. The distribution and morphological characteristics of the residual oil in the micro-etched glass model were observed and analyzed through photographs.

[0148] ImageJ software was used to perform quantitative analysis on the overall and local oil displacement images of the micro-model.

[0149] The macroscopic oil displacement effect of high-mineralized zinc oxide nanofluids was recorded using a camera, such as... Figure 8 As shown in Figure A.

[0150] ImageJ software was used to perform quantitative analysis on micro-etched glass models and localized images of high-mineralization zinc oxide nanofluid-driven oil recovery. Figure 8 As shown in B.

[0151] The permeability of the micro-etched glass model is less than 10 × 10⁻⁶. -3 μm 2This oil belongs to the ultra-low permeability category, with a complex pore structure and an interwoven distribution of pores and throats. After the first waterflood, crude oil in the larger pore channels is displaced, but clusters, sheets, and films of residual oil form at the pore throats, with films being the most prevalent. After displacement by high-mineralization zinc oxide nanofluid, the film-like residual oil is dispersed into droplets, increasing the oil phase dispersibility. After the second waterflood, the residual oil saturation decreases, and the recovery rate of high-mineralization zinc oxide nanofluid flooding increases by 40.39%, indicating that nanofluid flooding significantly improves the recovery rate (p < 0.01).

[0152] Example 20

[0153] Use emulsification index (E) 24 The emulsifying ability of the high-mineralization-resistant zinc oxide nanofluid long-chain hydrocarbon synthesized in Example 5 was determined.

[0154] The oil phase is simulated oil, and the water phase is simulated formation water. E 24 The specific operating steps for the determination are as follows:

[0155] Take 3 mL of high-mineralization-resistant zinc oxide nanofluid and mix it with kerosene, vegetable oil, liquid paraffin, n-heptane, and n-hexadecane at a volume ratio of 1:1. After thoroughly shaking and mixing, let it stand at room temperature for 24 h. Measure the height of the emulsion layer and the total height of the mixture, and calculate E according to the following formula. 24 value.

[0156]

[0157] Five substrates—kerosene, vegetable oil, liquid paraffin, n-heptane, and hexadecane—were used to evaluate the emulsifying activity of high-mineralization zinc oxide nanofluids. The emulsifying effects are shown in [Figure / Reference]. Figure 9 .

[0158] Depend on Figure 9 It can be seen that the high-mineralization-resistant zinc oxide nanofluid exhibits good emulsifying properties, and the high-mineralization-resistant zinc oxide nanofluid shows the best emulsifying effect on kerosene and hexadecane. 24 Reaching 58.1%; E for vegetable oils, liquid paraffin, and n-heptane 24The values ​​were 48.9%, 54.6%, and 55.4%, respectively. The high-mineralization-resistant zinc oxide nanofluid possesses a high specific surface area and surface energy, enabling it to adsorb at the oil-water interface, forming a stable and dense interfacial film, reducing interfacial tension, and thus enhancing emulsification. Simultaneously, the charged surface of the nanoparticles can form a charge layer around oil droplets, increasing the electrostatic repulsion between droplets and improving emulsion stability. The Halomonas sp. strain CDH-2 metabolites in the high-mineralization-resistant zinc oxide nanofluid synergistically form a composite film through hydrogen bonding / electrostatic interactions, improving interfacial stability, preventing oil droplet aggregation, and further increasing the emulsification index. Therefore, the high-mineralization-resistant zinc oxide nanofluid exhibits excellent hydrocarbon emulsification capabilities and can be used for oil well huff and puff.

[0159] Example 21

[0160] The viscosity-reducing ability of the high-mineralization zinc oxide nanofluid synthesized in Example 5 was tested using microscopic observation techniques and a viscometer.

[0161] The oil phase consisted of crude oil from Jianghan Oilfield and Shengli Oilfield, with viscosities of 2264 and 12900 mPa·s, respectively, at 40 ℃. The specific operating procedures for the determination are as follows:

[0162] High-mineralization resistant zinc oxide nanofluid was mixed with crude oil from Jianghan and Shengli oilfields at a mass ratio of 5:1 and cultured in a shaker at 40℃ and 170 r / min for 4 days to form an oil-water mixture.

[0163] Operate the viscometer according to the instructions and measure the viscosity of the above oil-water mixture. Repeat the measurement three times and take the average value. The viscosity of the above oil-water mixture is shown in Table 6.

[0164] Take 20 μL of the above mixture and observe the oil-water state under a microscope. The microscopic morphology of the oil-water mixture is shown in the figure. Figure 10 .

[0165] Table 6. Viscosity of oil-water mixtures after application to high-mineralization zinc oxide nanofluids

[0166]

[0167] As shown in Table 6, after the action of the high-mineralization zinc oxide nanofluid, the viscosity of the oil-water mixture of crude oil from Jianghan Oilfield and Shengli Oilfield decreased to 56.23 mPa·s and 89.12 mPa·s, respectively, with a viscosity reduction rate of >95%.

[0168] like Figure 10As shown in the image, under a microscope, the crude oil emulsion formed after treatment with high-mineralization-resistant zinc oxide nanofluid exhibits a dense number of small, uniformly distributed oil droplets, with the equivalent circular diameter of the droplets concentrated in the range of 2-15 μm, accounting for over 95% of the total. The CDH-2 metabolites of *Halomonas* sp. in the high-mineralization-resistant zinc oxide nanofluid can reduce the oil-water interfacial tension and induce initial emulsification. The zinc oxide nanoparticles in the high-mineralization-resistant zinc oxide nanofluid are then irreversibly adsorbed onto the oil droplet surface, forming a rigid particulate film that prevents droplet aggregation. Simultaneously, under shearing action, an O / W emulsion system is formed. Therefore, the high-mineralization-resistant zinc oxide nanofluid possesses excellent viscosity-reducing ability for heavy oil and can be used for heavy oil viscosity reduction.

[0169] In summary, the zinc oxide nanofluid synthesized by Halomonas mediated by this invention can effectively improve oil recovery. It can maintain good and stable dispersion in high-salinity water, while reducing oil-water interfacial tension, changing the wettability of oil-wetting surfaces, emulsifying and dispersing crude oil, and reducing crude oil viscosity. It has potential application prospects in the field of improving oil recovery in the oil and gas industry.

[0170] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A type of Halomonas CDH-2, characterized in that, The preservation number of the Halomonas sp. strain CDH-2 is CCTCC NO: M 2025786.

2. The Halomonas CDH-2 as described in claim 1, characterized in that, The 16S rRNA nucleotide sequence of the *Halomonas silicoides* CDH-2 is shown in SEQ ID NO.

1.

3. A fermentation broth, characterized in that, The fermentation broth is the fermentation broth of the Halomonas CDH-2 as described in claim 1 or 2.

4. The method for preparing fermentation broth as described in claim 3, characterized in that, Specifically, it includes: The *Haloxymonas halosa* CDH-2 of claim 1 is inoculated into a high-salt culture medium, fermented, and the fermentation broth is collected to obtain the product.

5. The method for preparing fermentation broth as described in claim 4, characterized in that, The high-salt culture medium comprises the following components: NaCl 20-200 g / L, glucose 25-35 g / L, NaNO3 8-12 g / L, Na3C6H5O7 2-4%, KH2PO4 0.5-1.5 g / L, (NH4)2HPO4 0.5-1.5 g / L, MgSO4·7H2O 0.1-0.3 g / L, and Na2MoO4 0.02-0.14 g / L.

6. The method for preparing fermentation broth as described in claim 4, characterized in that, The fermentation culture temperature is 35-40 ℃, and the fermentation culture time is 1-7 days.

7. A method for synthesizing highly mineralizable metal nanofluids mediated by Halomonas, characterized in that, Specifically, it includes: The fermentation broth of Halomonas CDH-2 described in claim 3 was mixed with a metal precursor solution and cultured to obtain a metal nanofluid resistant to high mineralization.

8. The method for synthesizing highly mineralized metal nanofluids as described in claim 7, characterized in that, The metal precursor solution has a metal ion molar concentration of 20-40 mM and a pH of 7.0-7.

2. The volume ratio of the metal precursor solution to the fermentation broth is 1:(1-1.5); The mixed culture temperature is 35-40 ℃; The mixed culture time is 12-48 h.

9. The high-mineralization-resistant metal nanofluid synthesized by the method described in claim 7.

10. The application of the high-mineralization-resistant metal nanofluid as described in claim 9 in any one of the following (I)-(III), characterized in that, (I) Oil displacement; (II) Oil well intake and output; (III) Reduce viscosity of heavy oil.

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