Shewanella resistant to multiple surfactants, bacterial agent and application thereof in thickened oil viscosity reduction

Abstract

The present application belongs to the technical field of microbial resource development and petroleum bioengineering, and particularly relates to a Shewanella producing surfactant with multiple tolerances, a bacterial agent and application of the Shewanella in thick oil viscosity reduction. Shewanella fodinae Specifically, the present application provides a Shewanella (S-11) producing phospholipid surfactant, a low-cost preparation method based on the strain, Bacillus Z-13 and Vibrio fluvialis with glycerol as a carbon source. The S-11 has the dual abilities of producing phospholipid surfactant and cracking long-chain alkanes, and the produced surfactant is a complex phospholipid system, with an emulsification index (E 24 ) stable at 55% or above under the conditions of pH 5-10, temperature 15-55 DEG C and high salinity. The three-strain complex fermentation process with glycerol as a carbon source can significantly reduce the production cost while maintaining excellent emulsification performance, thereby being suitable for thick oil exploitation, pipeline transportation, oil pollution remediation and other fields.

Description

Technical Field

[0001] This invention belongs to the field of microbial resource development and petroleum bioengineering technology, specifically involving a multi-tolerant Shewanella strain that produces surfactants, a bacterial agent, and its application in reducing viscosity of heavy oil. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In recent years, conventional oil and gas resources have become increasingly depleted, making heavy oil a strategic resource for global energy supply. Heavy oil is rich in long-chain hydrocarbons with high carbon numbers, exhibiting high internal friction, poor fluidity, and high viscosity. Developing microbial technologies with "chemical modification and component lightening" functions can improve the interfacial properties of heavy oil through microbial surfactants and directionally break down long-chain alkanes into short-chain molecules, achieving lightening and thus meeting broad market demand.

[0004] Currently, the microbial surfactants used for reducing viscosity in heavy oil are mainly glycolipids and lipopeptides. These form micellar interfacial films with low strength, which are prone to demulsification over long distances, leading to viscosity recovery and reduced recovery and transportation efficiency. Phospholipids (such as phosphatidylethanolamine) can self-assemble into vesicle structures, resulting in high interfacial film strength. However, existing phospholipid surfactants are mainly used for oil pollution remediation and lack the ability to break down and lighten long-chain alkanes in heavy oil, thus affecting flooding efficiency.

[0005] Furthermore, the high concentration and mineralization of calcium and magnesium in oil reservoirs easily lead to the inactivation of microorganisms and surfactants. Glycolipids readily complex and precipitate with calcium and magnesium in hard water, while lipopeptides tend to produce large amounts of foam, and Bacillus subtilis fermentation yields are extremely low. The purification of rhamnolipids produced by Pseudomonas is complex, and many are pathogenic bacteria (such as Pseudomonas aeruginosa), making it difficult to meet the requirements for large-scale engineered cultivation and ecological safety. Simultaneously, single-strain fermentation faces high metabolic pressure and limited product accumulation during industrial scale-up, making it difficult to overcome yield and performance bottlenecks. Summary of the Invention

[0006] This invention provides a wild-type Shewanella strain with multiple tolerances to phospholipid-producing surfactants, and a method for preparing surfactants based on this strain in combination with Bacillus Z-13 and Vibrio fluvialis, using inexpensive glycerol as a carbon source. This method can be used for viscosity reduction and quality improvement treatments in heavy oil production, transportation, and processing, and can also meet the needs of similar environmental surfactants in other fields. This invention is based on the above research results.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a strain of Shewanella, the strain of which is named Shewanella (… Shewanella fodinaeS-11, this strain was deposited on July 28, 2025 at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province), with accession number CCTCC NO: M 20251697.

[0008] This viscosity-reducing bacterium, *Shewanella*, is environmentally friendly and possesses the ability to produce phospholipid surfactants and decompose long-chain alkanes. The surfactant yield is approximately 50 g / L, exhibiting high activity. Both the strain and the surfactant demonstrate good tolerance to salinity, alkalinity, and high mineralization. The emulsification performance reaches approximately 55%, and the viscosity reduction rate for heavy oil at room temperature reaches 70%. Based on the verification of the excellent physicochemical properties of the surfactant produced by the S-11 single strain, and to meet the needs of industrial scale-up and replace expensive laboratory-grade culture media, a method for preparing a multi-strain compound surfactant based on this strain is further provided. The product emulsification index can reach 62%, significantly reducing raw material costs compared to the single-strain LB culture system.

[0009] A second aspect of the present invention provides a microbial inoculant, wherein the active ingredient of the microbial inoculant is (a) or (b): (a) The above-mentioned Shewanella bacteria; (b) The above-mentioned Shewanella, Bacillus Z-13 and Vibrio fluvialis.

[0010] A third aspect of the present invention provides a method for preparing the above-mentioned microbial inoculant, the method comprising: Strains S-11, Bacillus Z-13, and Vibrio fluvialis were co-inoculated in a medium with glycerol as the carbon source for aerobic fermentation.

[0011] In a fourth aspect, the present invention provides the use of the above-mentioned Shewanella bacteria or the above-mentioned microbial agents as or in the preparation of surfactants.

[0012] A fifth aspect of the present invention provides the use of the above-described Shewanella bacteria or the above-described microbial agent in any one or more of the following: a) Petroleum emulsification; b) Petroleum viscosity reduction treatment; c) Oil extraction and transportation; d) Oil pollution remediation; e) Petrochemical industry; f) Daily necessities and food.

[0013] Compared with existing technologies, the above technical solution has the following advantages: The above technical solution provides Shewanella ( Shewanella fodinaeIn the process of reducing viscosity in heavy oil, S-11 breaks through the traditional single viscosity reduction mode of microorganisms that rely solely on surfactant emulsification. It possesses a synergistic viscosity reduction mechanism of "oxidative cleavage of long-chain alkanes" and "emulsification and dispersion by biosurfactants." Gas chromatography analysis confirms that this strain can shear the long-chain alkane components in heavy oil that cause high viscosity, converting them into short-chain alkanes with better flowability, thus achieving a lightening modification of heavy oil components. At the same time, in conjunction with its secreted highly efficient surfactants, it forms a stable emulsion layer at the oil-water interface, enabling efficient viscosity reduction and quality improvement of heavy oil.

[0014] Whole-genome sequencing results indicate that strain S-11 is an environmentally friendly, non-pathogenic, wild-type oil reservoir strain that can grow and reproduce in situ within oil reservoirs, continuously producing surfactants through metabolism. This allows for "one-time application with long-term effectiveness," effectively addressing the technical challenge of viscosity-reducing effects easily rebounding in heavy oil extraction. It exhibits broad-spectrum environmental tolerance, maintaining vigorous metabolic activity and growth in environments with sodium chloride salinity of 0-6%, seawater salinity of 1-6%, pH of 5-10, and temperatures of 15-45℃.

[0015] The strain provided by the above technical solution possesses highly efficient surfactant synthesis capabilities, with liquid fermentation yields reaching up to 50 g / L, demonstrating significant potential for industrial-scale production. Component analysis shows that the produced surfactants not only contain complete double-chain phospholipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC), but are also rich in lysophospholipids (such as LPC and LPE) generated through bioenzyme modification, forming phospholipid complexes resembling cell membrane components. This phospholipid surfactant exhibits strong resistance to calcium and magnesium ion interference and stability in complex, highly mineralized environments: under conditions of pH 5-10, temperature 15-45℃, and high mineralization levels of 1-9% sodium chloride, 1-4% magnesium chloride, 1-5% calcium chloride, or 1-4% hydrochloric acid, the emulsification index (E) remains high. 24 It remains stable at around 57%.

[0016] The preparation of a multi-strain compound surfactant based on strain S-11 uses glycerol as the carbon source, replacing traditional nutrient-rich culture media with a cheap and renewable carbon source. While maintaining excellent emulsifying properties, it significantly reduces the production cost of surfactants, demonstrating good potential for industrial-scale application. The compound bacterial agent exhibits good emulsifying properties in environments with sodium chloride 1-7%, seaweed concentration 1-6%, and Mg... 2+ Concentration 1-200 mmol / L%, Ca 2+ Under concentration conditions of 1-150 mmol / L%, the emulsification index remains stable at over 50%, and can reach up to 62%. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 Photographs (A) and (B) show the growth of Shewanella S-11 on LB solid medium at room temperature.

[0019] Figure 2 Phylogenetic tree of Shewanella S-11 16S rDNA gene sequence.

[0020] Figure 3 The growth curves and emulsifying ability of strain S-11 at different NaCl concentrations are shown.

[0021] Figure 4 The growth curves and emulsifying ability of strain S-11 at different sea salt concentrations are shown.

[0022] Figure 5 The growth curves and emulsifying ability of strain S-11 at different pH values ​​are shown.

[0023] Figure 6 The growth curves and emulsifying ability of strain S-11 at different temperatures are shown.

[0024] Figure 7 The emulsifying ability of the extracted surfactant under different conditions.

[0025] Figure 8 FTIR spectral analysis of surfactants in Shewanella S-11.

[0026] Figure 9 This is a graph showing the change in viscosity of heavy oil after treatment with bacterial solution and surfactant at room temperature.

[0027] Figure 10 The results show the composition of petroleum hydrocarbon samples before and after inoculation with Shewanella S-11.

[0028] Figure 11 S-11 was streaked separately (A) and S-11 was co-streaked with strain 3 (B).

[0029] Figure 12 The emulsifying ability of compound bacterial agents under different conditions. Detailed Implementation

[0030] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0032] In a typical embodiment of the present invention, a strain of Shewanella is provided, which is named Shewanella. Shewanella fodinae S-11, this strain was deposited on July 28, 2025 at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province), with accession number CCTCC NO: M 20251697.

[0033] For ease of description, the Shewanella bacteria described in this invention Shewanella fodinae S-11 is also abbreviated as strain S-11 or S-11 in the application documents.

[0034] The strain S-11 of this invention can be cultured in LB medium. Single colonies are round with neat edges, pale yellow in color, and have a smooth surface.

[0035] Experiments have shown that S-11 can grow in sodium chloride concentrations ranging from 0% to 6%, exhibiting good salt tolerance. Furthermore, it can produce active substances at sodium chloride concentrations of 0% to 4%, achieving an emulsification index of up to 56%. S-11 can also grow and produce active substances at sea salt concentrations of 1% to 6%, achieving an emulsification index of 57%. S-11 can grow and produce active substances at pH levels of 5% to 10, achieving an emulsification index of up to 57%. Additionally, S-11 can grow and produce active substances at temperatures ranging from 15℃ to 45℃, demonstrating excellent emulsification effects, reaching up to 60%.

[0036] In another specific embodiment of the present invention, a microbial inoculant is provided, wherein the active ingredient of the microbial inoculant is (a) or (b): (a) The above-mentioned Shewanella bacteria; (b) The above-mentioned Shewanella, Bacillus Z-13 and Vibrio fluvialis.

[0037] The bacterial agent can be used as a surfactant.

[0038] In addition to the active ingredients, the microbial inoculant also contains a carrier. The carrier can be any biologically inert carrier commonly used in the field of microbial inoculants, and is not specifically limited here.

[0039] The Bacillus Z-13 strain, with accession number CCTCC NO: M 20211594, has been disclosed in Chinese patent CN 115960743 A. This strain possesses the ability to produce lipopeptide surfactants, and when combined with Shewanella S-11, it can synergistically improve the emulsifying properties and yield of surfactants.

[0040] Vibrio riverine ( Vibrio fluvialis Shewanella (Vibrio spp.) is a Gram-negative bacillus belonging to the genus Vibrio of the family Vibrioceae. This bacterium is straight or arc-shaped, possessing both apical and lateral flagella. This invention has revealed a synergistic symbiotic relationship between Shewanella and Vibrio fluvialis. Shewanella does not grow when inoculated alone in a medium with glycerol as the sole carbon source, but grows normally when co-cultured with Vibrio fluvialis, indicating a metabolic synergistic mechanism between the two. In a specific embodiment of this invention, Vibrio fluvialis is specifically Vibrio fluvialis ATCC 33809, which is the type strain of Vibrio fluvialis and is commercially available. In this invention, Vibrio fluvialis ATCC 33809 is named Vibrio fluvialis strain 3 or simply strain 3.

[0041] In another specific embodiment of the present invention, a method for preparing the above-mentioned microbial inoculant is provided, the method comprising: Strains S-11, Bacillus Z-13, and Vibrio fluvialis were co-inoculated into a glycerol-based carbon source medium for aerobic fermentation. The aerobic fermentation conditions were: cultured at 25-30℃ (preferably 28℃) and 160-200 r / min (preferably 180 r / min) for 12-120 h (preferably 48 h).

[0042] The culture medium consisted of: 15 ml / L glycerol, 1.5 g / L ammonium chloride, 4.8 g / L HEPES, 0.5 g / L potassium chloride, 0.02 g / L potassium dihydrogen phosphate, 0.02 g / L ferrous sulfate heptahydrate, and 0.3 g / L yeast extract.

[0043] In one specific embodiment of the present invention, the above-mentioned Shewanella bacteria or the above-mentioned compound surfactant can be used as a viscosity reducer, especially a petroleum viscosity reducer.

[0044] In one specific embodiment of the present invention, the application of the above-mentioned Shewanella or the above-mentioned microbial agent in any one or more of the following: a) Petroleum emulsification; b) Petroleum viscosity reduction treatment; c) Oil extraction and transportation; d) Oil pollution remediation; e) Petrochemical industry; f) Daily necessities and food.

[0045] In one specific embodiment of the present invention, the compound microbial agent can be applied in a saline-alkali and / or highly mineralized environment.

[0046] The saline-alkali and / or high-mineralization environment is characterized by sodium chloride concentration of 1-7%, hydrochloric acid concentration of 1-6%, and Mg concentration of 1-7%. 2+ Concentration 1-200 mmol / L, Ca 2+ Concentration 1-150 mmol / L.

[0047] In one specific embodiment of the present invention, a method for reducing oil viscosity is provided, the method comprising applying the aforementioned Shewanella bacteria or the aforementioned microbial agent to the oil. Specifically, by applying the Shewanella bacteria or the surfactant to crude oil in a pipeline, the viscosity of the crude oil can be reduced and its fluidity improved, thereby facilitating oil extraction and transportation.

[0048] In this invention, the microbial agent can reduce viscosity through the following synergistic mechanism: a) The Shewanella bacteria metabolizes and oxidizes long-chain alkanes in heavy oil, converting them into short-chain alkanes, thereby achieving in-situ lightening of heavy oil components; b) The phospholipid surfactant forms a stable emulsion layer at the oil-water interface, reduces interfacial tension, and improves the permeability of crude oil in porous media. c) The Shewanella bacteria can grow and reproduce in situ in the reservoir, continuously secreting surfactants to achieve long-term viscosity reduction.

[0049] In one specific embodiment of the present invention, a method for treating and / or remediating a petroleum-polluted environment is provided, the method comprising applying the aforementioned Shewanella bacteria or the aforementioned microbial agent to the petroleum-polluted environment.

[0050] In one specific embodiment of the present invention, the petroleum-polluted environment can be petroleum-polluted water bodies and petroleum-polluted soil. Applying the *Shewanella* bacteria or the surfactant to the petroleum-polluted water bodies or soils promotes further biodegradation of petroleum hydrocarbons by oxidatively breaking down long-chain alkanes to degrade pollutants.

[0051] In this invention, the petroleum can specifically be heavy oil.

[0052] In this invention, the above-mentioned Shewanella bacteria or microbial agents are used as surfactants due to their non-toxicity, strong biocompatibility, and excellent emulsifying properties. They are used as emulsifiers, dispersants, or surfactants, and can be applied to daily necessities (including daily chemical products, further including toiletries, cosmetics, etc.) and food. No specific limitations are made here.

[0053] Example 1: Isolation and screening of Shewanella S-11 Step X1: Take 100 μL of water sample from the drain well and spread it evenly onto LB solid medium plates using the plate spreading method for incubation. The LB solid medium consisted of 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar. It was sterilized in an autoclave at 121°C for 20 minutes and then enriched in a constant temperature shaking incubator at 28°C and 120 r / min for 48 hours.

[0054] Step X2: Pick colonies of different morphologies from the solid culture medium after X1 culture using an inoculation loop, and inoculate them onto LB solid culture medium using the streak plate method. Incubate them in a constant temperature incubator at 30℃ and 120r / min for 48h. After the colonies grow, pick single colony clones and inoculate them onto LB solid culture medium in the same way. Each strain with the same morphology was inoculated into three solid culture media for parallel experiments.

[0055] Step X3: Repeat the separation process of step X2 until purified colonies with a single morphology are obtained.

[0056] The purified colonies can be expanded in LB liquid medium and stored at 4°C for immediate use.

[0057] like Figure 1 As shown, when the strain grows on LB solid medium, single colonies are round with neat edges, a light yellow color, and a smooth surface.

[0058] 16S rDNA sequence analysis of the isolated strains and sequence alignment in NCBI showed that the Sheva strains screened in this invention are... Shewanella fodinae It was named S-11. A phylogenetic tree was constructed using MEGA software with the Neighbor Joining method, as follows: Figure 2 As shown.

[0059] The preservation method is to mix 1 ml of bacterial culture with 0.5 ml of 30% glycerol, place the mixture in a 1.5 ml centrifuge tube, seal it tightly with sealing film, and store it in a refrigerator at -80℃.

[0060] 16S rDNA sequence of the strain: Example 2: Determination of Surfactant Production Capacity of Shewanella S-11 Step X1: Inoculate the strain stored at -80℃ into LB liquid medium, activate and culture it, and use the cultured strain as a seed culture. The LB liquid culture medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride. It was sterilized in an autoclave at 121°C for 20 minutes and then cultured in a constant temperature shaking incubator at 28°C and 180 r / min for 48 hours.

[0061] Step X2: Seed cultures were inoculated onto LB liquid medium and cultured under different salinity, pH, and temperature conditions, with shaking for 2 days at a shaking speed of 180 r / min. The culture medium was collected at set time points to determine the growth capacity of the strain and the level of biosurfactant production under different conditions.

[0062] The determination of the biosurfactant production capacity of the bacterial solution includes: evaluating the ability of the strain to produce surfactant using the emulsification index; adding 3 ml of kerosene and 3 ml of bacterial solution to a clean test tube, shaking the test tube vigorously for 2 minutes to mix thoroughly, allowing it to stand at room temperature for 24 hours, and then measuring the ratio of the emulsion layer to the total liquid height; calculating the emulsification index using the following formula: Emulsification index = (Emulsion layer height / Total liquid height) × 100% The set time is 0 h, 6 h, 12 h, 24 h, 30 h, 36 h, and 48 h; the NaCl gradient is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%; the seawater concentration is 0%, 1%, 2%, 3%, 4%, 5%, and 6%; the pH gradient is 5, 6, 7, 8, 9, and 10; and the temperature gradient is 15 ℃, 25 ℃, 35 ℃, 45 ℃, and 55 ℃.

[0063] from Figure 3-6 The growth and emulsifying abilities of Shewanella S-11 under different salinity, alkalinity, and temperature conditions were obtained. S-11 can grow in NaCl concentrations ranging from 0% to 6%, exhibiting good salt tolerance. It can produce active substances at sodium chloride concentrations of 0% to 4%, with an emulsification index reaching 56%. S-11 can grow in seawater concentrations of 1% to 6% and has excellent emulsifying properties. S-11 can grow and produce active substances in a pH range of 5% to 10, with an emulsification index reaching 57%. S-11 can grow and produce active substances in a temperature range of 15℃ to 45℃, with excellent emulsification effects, reaching 60%.

[0064] Example 3: Identification of Shewanella's tolerance to S-11 surfactant Step X1: Inoculate the strain stored at -80℃ into LB liquid medium, activate and culture it, and use the cultured strain as a seed culture. Step X2: Seed cultures were inoculated onto LB liquid medium and shaken at 28°C for 2 days at a shaking speed of 180 r / min. After 2 days, the culture medium was collected to extract surfactants for surfactant tolerance testing.

[0065] The method for determining the surfactant tolerance includes: evaluating the emulsifying ability of the surfactant using the emulsification index; adding 3 ml of kerosene and 0.1 ml of surfactant mixed with 3 ml of the appropriate solution to a clean test tube; vigorously shaking the test tube for 2 min to ensure uniform mixing; allowing it to stand for 24 h under appropriate conditions; measuring the ratio of the emulsion layer to the total liquid height; and calculating the emulsification index using the following formula: Emulsification index = (Emulsion layer height / Total liquid height) × 100% The pH gradient is 5, 6, 7, 8, 9, 10; the temperature gradient is 15℃, 25℃, 35℃, 45℃, 55℃; the NaCl gradient is 1%, 3%, 5%, 7%, 9%; the hydroxyl gradient is 1%, 2%, 3%, 4%, 5%; the MgCl2 gradient is 1%, 2%, 3%, 4%, 5%; and the CaCl2 gradient is 1%, 2%, 3%, 4%, 5%.

[0066] from Figure 7 It can be seen that surfactants can exert good effects in pH 5-10, temperature 15-45℃, NaCl 1-9%, hydrochloric acid 1-4%, MgCl2 1-4%, and CaCl2 1-5%.

[0067] Example 4: Identification and yield of surfactants in Shewanella S-11 Step X1: Inoculate the preserved strain into LB liquid medium, activate and culture it, and use the cultured strain as a seed culture.

[0068] Step X2: Inoculate the seed culture into 1L LB medium and culture at 28 ℃ and 180 r / min for 48 h. Centrifuge the bacterial suspension (8000×g, 10 min) to collect the bacterial cells, resuspend them in physiological saline, and sonicate them. Mix the broken cell suspension with methanol:chloroform = 2:1 v / v, shake thoroughly, take the lower chloroform phase, and rotary evaporate it at 40 ℃ to constant weight to obtain the crude surfactant extract.

[0069] Step X3 involves performing infrared spectroscopy and liquid chromatography-mass spectrometry analysis on the extracted surfactant.

[0070] After rotary evaporation, approximately 50g of crude liquid surfactant was obtained, which has extremely strong emulsifying properties. Figure 8 The FTIR spectral analysis of the surfactant is presented, allowing identification of the functional groups present in the surfactant. The broad peak at 3382 cm⁻¹ indicates the presence of hydroxyl and amino groups. The distinct absorption peak at 1723 cm⁻¹ corresponds to the C=O stretching vibration of the ester group, while the absorption peak at 1654 cm⁻¹ can be attributed to the C=C stretching vibration or the Amide I (amide C=O) vibration region of the protein / peptide chain. The vibration at 1534 cm⁻¹ can be attributed to the aromatic ring skeletal vibration or the Amide II (NH bending / CN stretching) vibration of the protein / peptide chain, and is similar to that at 1650 cm⁻¹. - The co-occurrence of peaks ¹ suggests the presence of peptide bonds or protein residues in the sample. The absorption peak at 1398 cm⁻¹ often corresponds to aliphatic CH deformation vibrations or carboxylates (COO₂). - Symmetric stretching. The absorption peak at 1232 cm⁻¹ indicates phosphate ester PO₂. - Antisymmetric stretching or CO (ester bond) stretching vibration is one of the typical characteristic peaks of phospholipid compounds. The peak at 1048 cm⁻¹ is attributed to CO or POC stretching vibrations in ester bonds. Based on the above characteristic peaks, it is inferred that this surfactant is a phospholipid surfactant.

[0071] The surfactant was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the collected data were imported into the LipidMAPS structural database (LMSD) for retrieval and comparison. Through exact mass matching and isotope abundance analysis, combined with fragmentation characteristics from secondary mass spectrometry (MS / MS), it was identified as a complex lipid system derived from cell membrane components. As shown in Table 1, component analysis revealed that this system not only contains intact double-chain phospholipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC), but is also rich in lysophospholipids (such as LPC and LPE) generated through bioenzyme modification. Lysophospholipids are responsible for rapid oil washing and viscosity reduction, while intact phospholipids are responsible for maintaining stability and preventing demulsification. The natural ratio of these two components is the core reason why this system achieves efficient viscosity reduction in heavy oils and is less prone to stratification.

[0072] Table 1 Results of surfactant analysis by liquid chromatography-mass spectrometry

[0073] Example 5: Determination of viscosity-reducing ability Step X1: Inoculate the preserved strain into LB liquid medium and activate it for 48 hours to use as a seed culture. Step X2: The seed culture was inoculated into LB medium and cultured for 48 h. The surfactant was extracted according to the method described in Step X2 of Example 4. An equal volume of bacterial solution (50 ml) and 2.5% (v / v) crude surfactant were added to the heavy oil. The oil was cultured at 25°C and 180 r / min for 3 d, 7 d, and 14 d, respectively. The viscosity of the heavy oil was measured using an LVDV-2T touch screen viscometer with a No. 25 rotor.

[0074] Step X3: Take strain S-11 to treat petroleum hydrocarbon samples, take untreated and treated petroleum hydrocarbons, dilute them 1000 times, and perform gas chromatography tests.

[0075] Experimental results are as follows Figure 9 As shown, strain S-11 reduced the viscosity of heavy oil with an initial viscosity of 15000 cp to 10875 cp after 3 days, to 7925 cp after 7 days, and to 4589 cp after 14 days, with a viscosity reduction rate of approximately 70%, demonstrating a good and sustained viscosity-reducing effect. The treatment group with direct addition of surfactant showed even faster viscosity reduction, with the viscosity decreasing to approximately 5789 cp after 7 days. The results are as follows... Figure 10 As shown, GC analysis indicates that long-chain alkanes are converted to medium- and short-chain alkanes after the strain's action, further verifying the component lightening mechanism.

[0076] Example 6: Preparation and Emulsification Performance Verification of Compound Microbial Agent Examples 1 to 5 above have sufficiently qualitatively verified the molecular structure of the phospholipid surfactants produced by the S-11 single strain and their stability under extreme environments. This example provides a low-cost compound preparation process based on inexpensive carbon source glycerol.

[0077] Step X1: The preserved strains S-11, Z-13 and strain 3 were inoculated into LB medium and activated at 28℃ and 180 r / min for 48 h, respectively, and used as seed cultures.

[0078] The strain S-11 is a wild Shewanella strain screened from reservoir produced water in this invention. Shewanella fodinae The phospholipid surfactant produced by this product has the accession number CCTCC NO: M 20251697 and its viscosity-reducing properties are detailed in Examples 1-5.

[0079] The strain Z-13 is Bacillus ( ) Bacillus sp. Z-13, which was patented on July 15, 2022 (CN 115960743 A), with accession number CCTCC NO: M 20211594, has the ability to produce lipopeptide surfactants. When combined with strain S-11, it can synergistically improve the emulsifying performance and yield of surfactants.

[0080] Bacterium No. 3 is Vibrio fluvialis ATCC 33809. This invention demonstrates through experiments that Bacterium No. 3 and S-11 have a synergistic symbiotic relationship. For example... Figure 11 As shown, the growth promotion experiment results indicate that S-11 does not grow when inoculated alone in a medium with glycerol as the sole carbon source, but can grow normally in the same medium when co-cultured with strain 3, verifying the metabolic synergy mechanism between the two.

[0081] Step X2: Activated strains S-11, Z-13, and strain 3 were centrifuged (8000×g, 10 min, repeated 3 times) to collect bacterial cells. The cells were resuspended in sterile physiological saline and washed to remove residual culture medium. They were then co-inoculated into glycerol medium at a ratio of S-11:Z-13:3 (v / v) of 4:3:3 (v / v) and cultured at 180 r / min for 48 h under different salinity and temperature conditions. The culture medium was collected at set time points to determine the production levels of biosurfactants under different conditions.

[0082] The glycerol culture medium consisted of 15 ml / L glycerol, 1.5 g / L ammonium chloride, 4.8 g / L HEPES, 0.5 g / L potassium chloride, 0.02 g / L potassium dihydrogen phosphate, 0.02 g / L ferrous sulfate heptahydrate, and 0.3 g / L yeast extract. The HEPES was sterilized by filtration through a 0.22 μm filter membrane. The solid culture medium contained 15 g / L agar.

[0083] The method for determining the emulsification index is the same as in Example 2.

[0084] The set time is 0 h, 6 h, 12 h, 24 h, 30 h, 36 h, 48 h; the NaCl gradient is 1%, 3%, 5%, 7%, 9%; the seawater concentration is 1%, 2%, 3%, 4%, 5%, 6%; the Ca... 2+ The concentrations were 1 mmol / L, 10 mmol / L, 50 mmol / L, 100 mmol / L, and 150 mmol / L; the Mg... 2+ The concentrations were 1 mmol / L, 5 mmol / L, 50 mmol / L, 100 mmol / L, and 200 mmol / L; the temperature gradients were 15 ℃, 25 ℃, 35 ℃, 45 ℃, and 55 ℃.

[0085] Figure 12 It can be concluded that the compound bacterial agent has good emulsification properties, with sodium chloride concentration of 1-7%, sea salt concentration of 1-6%, and Mg... 2+ Concentration 1-200 mmol / L, Ca 2+ Under concentrations of 1-150 mmol / L, the emulsification index remains stable at over 50%, reaching a maximum of 62%.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Shewanella, characterized in that, The Shewanella bacteria was named Shewanella. Shewanella fodinae S-11, this strain was deposited at the China Center for Type Culture Collection on July 28, 2025, with accession number CCTCC NO: M20251697.

2. A microbial inoculant, characterized in that, The active ingredient of the microbial agent is (a) or (b): (a) The Shewanella bacteria as claimed in claim 1; (b) The Shewanella, Bacillus Z-13 and Vibrio fluvialis as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The Vibrio fluvialis is Vibrio fluvialis ATCC33809.

4. The microbial agent as described in claim 2, characterized in that, The microbial agent contains a carrier.

5. The method for preparing the microbial inoculant according to any one of claims 2-4, characterized in that, The preparation method includes: Shewanella, Bacillus Z-13, and Vibrio fluvialis were co-inoculated into a medium with glycerol as the carbon source for aerobic fermentation.

6. The preparation method according to claim 5, characterized in that, The aerobic fermentation conditions are: cultured at 25-30℃ and 160-200 r / min for 12-120 h.

7. The use of Shewanella as described in claim 1 or the microbial agent as described in any one of claims 2-4 as a surfactant.

8. The application as described in claim 7, characterized in that, The surfactant is a phospholipid surfactant.

9. The use of Shewanella as described in claim 1 or the microbial agent as described in any one of claims 2-4 in any one or more of the following: a) Petroleum emulsification; b) Petroleum viscosity reduction treatment; c) Oil extraction and transportation; d) Oil pollution remediation; e) Petrochemical industry; f) Daily necessities and food.

10. The application as described in claim 9, characterized in that, The application environment is a saline-alkali and / or highly mineralized environment; Further, the saline and / or high salinity environment is 1-7% sodium chloride, 1-6% seawater concentration, Mg 2+ concentration 1-200 mmol / L, Ca 2+ concentration 1-150 mmol / L.

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