Aeromonas hydrophila D15 and application thereof in mercury pollution remediation

Abstract

The application discloses an aeromonas hydrophila D15 and application thereof in mercury pollution remediation, and belongs to the technical field of microbial application. The strain has been preserved in the China Center for Type Culture Collection on September 8, 2025, and the preservation number is CCTCC NO: M20251975. The strain is oval and fusiform, has mercury tolerance, and can mediate Hg 2+ / Se 4+ in the environment added with Hg 2+ and Se 4+ , and can synthesize mercury selenide nanoparticles outside cells. The particle size of the generated HgSe NPs is about 10 nm, and the characteristic diffraction peak of HgSe can be detected. Thus, the mercury pollution environment can be efficiently remediated, a new thought and scheme are provided for environmental governance and remediation, and the application has a wide application prospect and high economic value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and applied technology, specifically relating to a strain of Aeromonas hydrophila D15 and its application in mercury pollution remediation. Background Technology

[0002] Mercury is a highly toxic, bioaccumulative, and long-distance migrating heavy metal. Mercury pollution mainly originates from industrial production, mining, and agricultural inputs, and it exists in the environment primarily as Hg. 2+ It exists in various forms, is easily absorbed by organisms and accumulates through the food chain, causing serious harm to ecosystems and human health.

[0003] Currently, mercury pollution remediation technologies mainly include physical remediation, chemical remediation, and bioremediation. While physical and chemical remediation technologies can rapidly remove mercury, they suffer from high costs, complex operations, a tendency to cause secondary pollution, and difficulty in remediating low-concentration mercury pollution. Bioremediation, due to its advantages such as low cost, environmental friendliness, absence of secondary pollution, and applicability to large-scale in-situ remediation, has become a research hotspot for mercury pollution remediation.

[0004] Microbial remediation is a core approach to bioremediation, and utilizing the mercury tolerance and transformation capabilities of microorganisms to detoxify or fix mercury is a key direction. Selenium has a strong affinity for mercury and can reduce the bioavailability and toxicity of mercury through antagonistic effects. Utilizing microorganisms to mediate the binding of selenium and mercury to generate stable mercury selenide nanoparticles is a novel and efficient strategy for mercury pollution remediation. Among the mercury-tolerant microorganisms currently discovered, few strains can efficiently mediate the binding of selenium and mercury to generate extracellular mercury selenide nanoparticles. Therefore, screening for highly efficient strains with this characteristic is of significant practical importance for promoting the industrial application of mercury pollution bioremediation technologies. However, Aeromonas hydrophila strains with highly efficient mercury remediation capabilities have not yet been reported. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to propose a *Aeromonas hydrophila* strain with highly efficient ability to remediate mercury pollution and its application in mercury pollution remediation.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] This invention proposes a strain of Aeromonas hydrophila ( 嗜水气单胞菌 D15, accession number CCTCCNO: M20251975.

[0008] This strain was deposited at the China Center for Type Culture Collection on September 8, 2025, with accession number CCTCCNO: M20251975.

[0009] The 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0010] The present invention also proposes the above-mentioned Aeromonas hydrophila ( 嗜水气单胞菌 Fermentation broth or bacterial suspension of D15.

[0011] Preferably, the fermentation broth refers to some or all of the substances contained in the culture medium for culturing the strain, regardless of the form of the fermentation broth, including substances such as metabolites or secretions of the strain culture products, and the strain itself is also contained in the fermentation broth.

[0012] Preferably, the bacterial suspension refers to a suspension obtained by collecting bacterial cells using centrifugal force and uniformly dispersing the bacterial cells in water.

[0013] The present invention also proposes the above-mentioned Aeromonas hydrophila ( 嗜水气单胞菌 D15. Application of its fermentation broth or bacterial suspension in mercury pollution remediation.

[0014] Preferably, the application specifically includes the following steps: in a mercury-contaminated environment, adding selenium and the aforementioned Aeromonas hydrophila (… 嗜水气单胞菌 D15, its fermentation broth or bacterial suspension is sufficient.

[0015] Preferably, the mercury is Hg. 2+ The selenium mentioned is Se. 4+ .

[0016] The present invention also proposes the above-mentioned Aeromonas hydrophila ( 嗜水气单胞菌 D15. Application of its fermentation broth or bacterial suspension in the preparation of mercury pollution remediation reagents.

[0017] This invention also proposes a mercury pollution remediation agent, the main active ingredient of which includes the aforementioned Aeromonas hydrophila (…). 嗜水气单胞菌 D15, its fermentation broth or bacterial suspension.

[0018] Preferably, the above-mentioned Aeromonas hydrophila ( 嗜水气单胞菌 D15, its fermentation broth or bacterial suspension is the sole active ingredient.

[0019] This invention also proposes a method for preparing mercury selenide nanoparticles, comprising the following steps: in a solution containing Hg 2+ and Se 4+ The above-mentioned Aeromonas hydrophila ( ) was added to the solution. 嗜水气单胞菌 D15, its fermentation broth or bacterial suspension, is obtained.

[0020] Preferred, Hg 2+ The solution includes, but is not limited to, one or more of mercuric nitrate, mercuric chloride, mercuric acetate, and mercuric sulfate. Mercuric chloride is more preferably preferred.

[0021] Preferred, Se 4+The solution includes, but is not limited to, one or more of sodium selenite, sodium biselenite, and selenite. Sodium selenite is more preferably preferred.

[0022] Preferred, Hg 2+ The concentration of the solution is 20~100μM, more preferably 30~80μM, and even more preferably 50μM.

[0023] Preferred, Se 4+ The concentration of the solution is 20~100μM, more preferably 30~80μM, and even more preferably 50μM.

[0024] Preferably, the Aeromonas hydrophila ( 嗜水气单胞菌 The final concentration of the fermentation broth or bacterial suspension of D15 is 10. 5 -10 8 CFU / mL, more preferably 10 6 CFU / mL.

[0025] The beneficial effects of this invention are as follows: This invention is the first to propose a *Aeromonas hydrophila* strain with highly efficient ability to remediate mercury pollution. 气单胞菌属 嗜水的 D15, which has mercury tolerance, can be added with Hg 2+ / Se 4+ In this environment, the bacterial cell surface shrinks, which can mediate Hg. 2+ with Se 4+ The extracellular synthesis of mercury selenide nanoparticles enables efficient remediation of mercury-polluted environments, providing new ideas and solutions for environmental governance and remediation, with broad application prospects and high economic value.

[0026] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0027] Figure 1 The blank control group (A) and Hg in Example 2 of this invention 2+ +Se 4+ Group (B) contains 气单胞菌属 嗜水的 Comparison chart of changes in D15 culture medium; Figure 2 The blank control group and Hg in Example 2 of this invention 2+ +Se 4+ Group 嗜水气单胞菌 Scanning electron microscope (SEM) image (A) and transmission electron microscope (TEM) image (B) of D15 cells and particles. Figure 3 In Embodiment 2 of the present invention 嗜水气单胞菌 D15 in Hg2+ +Se 4+ The transmission electron microscope image (A) and the accompanying energy dispersive spectroscopy (EDS) surface scan (scale bar 500 nm / 200 nm) in the group are, in order, the Hg elemental distribution (B), the Se elemental distribution (C), and the superimposed Hg+Se elemental distribution (D).

[0028] Figure 4 In Embodiment 2 of the present invention 嗜水气单胞菌 D15 in Hg 2+ +Se 4+ The elemental distribution map and TEM image of the black substance separated from the group; where A is the transmission electron microscope image of the black substance, BD are the Hg elemental distribution, Se elemental distribution and Hg+Se elemental superposition distribution map of the black substance respectively, and E is the TEM image of the black substance (scale bar 25 nm / 5 nm): nanoparticles with a particle size of about 10 nm can be observed, and the interplanar spacing d = 0.356 nm; the characteristic (111) crystal plane of HgSe nanoparticles.

[0029] Figure 5 In Embodiment 2 of the present invention 嗜水气单胞菌 D15 in Hg 2+ +Se 4+ The XRD patterns in the group, with the horizontal axis representing 2θ (degree) and the vertical axis representing diffraction intensity, showed characteristic peaks (111), (200), (220), and (311) of HgSe (PDF#73-1668) in the range of 10 to 60°.

[0030] Figure 6 This is a photograph of the Aeromonas hydrophila D15 strain of this invention.

[0031] Figure 7 For the present invention 嗜水气单胞菌 The phylogenetic tree of D15. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0033] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0034] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0035] Example 1: Aeromonas hydrophila 嗜水气单胞菌 Isolation, identification and culture of D15 1. Culture medium: LB broth liquid medium and LB agar solid medium were used, with the following components: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L. The pH was adjusted to 7.0 ± 0.1 (25℃), and the medium was sterilized at 121℃ for 30 min before use.

[0036] 2. Strain isolation: Samples were collected from mercury-contaminated areas (such as mercury mines, mercury-contaminated farmland / water bodies), and added to LB liquid medium. Mercuric chloride was added to adjust the Hg² concentration in the medium. + The culture was carried out at a concentration of 50 μM in a shaker at 25℃ and 180 r / min. After serial dilution, plating on LB agar plates, and repeated streak purification, single colonies were obtained and identified as Aeromonas hydrophila. 嗜水气单胞菌, It was named D15. A photograph of this strain is shown below. Figure 6 As shown, the phylogenetic tree of this strain is as follows: Figure 7 As shown. This strain was deposited at the China Center for Type Culture Collection on September 8, 2025, with accession number CCTCCNO: M20251975.

[0037] The 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0038] 3. Strain culture: Pick a single colony and inoculate it into LB liquid medium. Incubate at 25℃ and 180 r / min in a shaker until the logarithmic growth phase. 4. Strain preservation: Add 0.3~0.5 mL of sterile glycerol to a 2 mL cryovial, then add 1 mL of bacterial suspension in the logarithmic growth phase, mix well, and let stand at room temperature for 30 min, then freeze at -80℃.

[0039] Example 2: Aeromonas hydrophila 嗜水气单胞菌 Mercury tolerance and selenium antagonism verification experiment of D15 1. Preparation of experimental reagents: 50 μM sodium selenite (Na2SeO3) solution: Weigh an appropriate amount of sodium selenite powder, dissolve it in LB liquid medium, filter it through a 0.22 μm filter membrane for sterilization, and set aside for use; 50 μM mercuric chloride (HgCl2) solution: Weigh an appropriate amount of mercuric chloride powder, dissolve it in LB liquid medium, filter it through a 0.22 μm filter membrane for sterilization, and set aside for use; 2. Experimental Groups: Four experimental groups were set up, namely... Control (blank control group): LB medium only; Se 4+ Group: LB + 50 μM Na2SeO3; Hg 2+ Group: LB + 50 μM HgCl2; Hg 2+ +Se 4+ Group: LB+50 μM HgCl2+50 μM Na2SeO3; Each group has 3 repetitions; 3. Inoculation and culture of bacterial strains: Strains in the logarithmic growth phase were inoculated into each experimental group. 嗜水气单胞菌 D15 bacterial suspension, to achieve a final bacterial concentration of 10. 6 CFU / mL, cultured on a shaker at 25℃ and 180 r / min; 4. Observation of cell morphology and nanoparticles: After a certain period of cultivation, samples were taken and the cell morphology and surrounding particle distribution were observed using scanning electron microscopy (SEM). The results showed that: 嗜水气单胞菌 D15 cells are oval and spindle-shaped, Hg 2+ +Se 4+ In one group, the bacterial cell surface was wrinkled and surrounded by nano-sized particles, while no obvious particles were generated in the other groups. 5. Characterization of mercury selenide nanoparticles: Elemental distribution detection: Hg was analyzed using an energy dispersive spectroscopy (EDS) instrument. 2+ +Se 4+ Elemental analysis of the bacterial cells and surrounding particles in the group revealed characteristic signals of Hg and Se in the extracellular environment, proving that Hg and Se are enriched in the extracellular environment. X-ray diffraction (XRD) detection: for Hg 2+ +Se 4+ The black precipitate generated by the group was detected by XRD, and the characteristic peaks (111), (200), (220), and (311) of HgSe (PDF#73-1668) were detected, proving that the precipitate was mercury selenide nanoparticles; Particle size analysis: Observation by transmission electron microscopy (TEM) showed that the generated HgSe NPs had a particle size of approximately 10 nm and a crystal interplanar spacing of d = 3.556 nm.

[0040] Aeromonas hydrophila 嗜水气单胞菌 D15, this strain is oval and spindle-shaped, and has mercury tolerance. (The sentence fragment about Hg appears unrelated and likely refers to a different strain.) 2+ / Se4+ In this environment, the bacterial cell surface shrinks, which can mediate Hg. 2+ with Se 4+ Mercuric selenide nanoparticles (HgSe NPs) were synthesized extracellularly. The generated HgSe NPs had a particle size of approximately 10 nm and the characteristic diffraction peaks of HgSe could be detected.

[0041] like Figure 1 As can be seen from this, a total of Hg was added. 2+ +Se 4+ The culture system of the treatment group produced a black precipitate, which was initially identified as HgSe nanoparticles.

[0042] like Figure 2 As can be seen from the blank control group 嗜水气单胞菌 D15 cells are typically oval and spindle-shaped; Hg was added. 2+ +Se 4+ The bacterial cells in the treatment group showed wrinkling and deformation on the surface, and a black precipitate was produced in the culture system. The black product was initially identified as HgSe nanoparticles. At the same time, a large number of nanoparticles were observed to be attached and distributed in the extracellular region of the bacterial cells.

[0043] like Figure 3 As shown, the elemental distribution spectra obtained by transmission electron microscopy (scale bar 500 nm / 200 nm) and its accompanying energy dispersive spectroscopy (EDS) surface scan show that: Hg 2+ +Se 4+ The extracellular regions of the co-cultured bacteria simultaneously detected signals of both Hg and Se, confirming that the biosynthesis of HgSe nanoparticles mainly occurs extracellularly within the bacterial strain; the product is primarily synthesized in situ extracellularly.

[0044] like Figure 4 As can be seen, the average particle size of the bio-derived HgSe nanoparticles is approximately 10 nm. Elemental analysis indicates that the substance is composed of Hg and Se. The interplanar spacing d = 0.356 nm corresponds to the (111) crystal plane of crystalline HgSe, confirming that the substance is HgSe. like Figure 5 As can be seen from the X-ray diffraction pattern (2θ: 10°~60°), the co-stressed sample shows characteristic diffraction peaks that match the HgSe standard PDF card (#73-1668), corresponding to the (111), (200), (220), and (311) crystal planes of HgSe, respectively, which confirms from a crystallographic perspective that the black precipitate is crystalline HgSe. SEQ ID NO.1: The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Aeromonas hydrophila ( Aeromonas hydrophila D15, characterized in that, The preservation number is CCTCC NO: M20251975.

2. The Aeromonas hydrophila described in claim 1 ( Aeromonas hydrophila Fermentation broth or bacterial suspension of D15.

3. The Aeromonas hydrophila described in claim 1 ( Aeromonas hydrophila D15. Application of the fermentation broth or bacterial suspension as described in claim 2 in the remediation of mercury pollution.

4. The application according to claim 3, characterized in that, The application specifically includes the following steps: In a mercury-contaminated environment, selenium and Aeromonas hydrophila (… Aeromonas hydrophila D15, its fermentation broth or bacterial suspension is sufficient.

5. The Aeromonas hydrophila as described in claim 1 ( Aeromonas hydrophila D15. Application of the fermentation broth or bacterial suspension according to claim 2 in the preparation of mercury pollution remediation reagents.

6. A mercury pollution remediation reagent, characterized in that, Its main active ingredient includes Aeromonas hydrophila as described in claim 1 ( Aeromonas hydrophila D15. The fermentation broth or bacterial suspension as described in claim 2.

7. The mercury pollution remediation reagent according to claim 6, characterized in that, Aeromonas hydrophila ( Aeromonas hydrophila D15, its fermentation broth or bacterial suspension is the sole active ingredient.

8. A method for preparing mercury selenide nanoparticles, characterized in that, In the presence of Hg 2+ and Se 4+ The solution was supplemented with Aeromonas hydrophila as described in claim 1. Aeromonas hydrophila D15, the fermentation broth or bacterial suspension as described in claim 2, is obtained.

9. The preparation method according to claim 8, characterized in that, Hg 2+ The concentration of the solution is 20~100μM; Se 4+ The concentration of the solution is 20~100μM.

10. The preparation method according to claim 8, characterized in that, The Aeromonas hydrophila ( Aeromonas hydrophila The final concentration of the fermentation broth or bacterial suspension of D15 is 10. 5 -10 8 CFU / mL.

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