Haloterrigena salina and application thereof

Abstract

The present application relates to a kind of halophilic halophanum and its application.The strain name is halophilic halophanum ( Salinicola halophilus )A3, is preserved in China Microorganism Strain Preservation Management Committee (CGMCC), and the preservation address is China Beijing City Chaoyang District Beichen West Road No. 1 Yard No. 3 Institute of Microbiology, Chinese Academy of Sciences, and the preservation date is July 30, 2025, and the preservation number is CGMCC No.35460.The halophilic halophanum ( Salinicola halophilus ) provided by the present application can be used for the degradation of toluene in high-salt environment.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology, specifically relating to a halophilic bacterium and its applications. Background Technology

[0002] Toluene, a typical volatile organic pollutant, is widely present in industrial processes such as pesticide synthesis, dye manufacturing, and petrochemical refining. These processes generate high-salt wastewater containing toluene, and the treatment of this industrial wastewater remains a persistent challenge due to the dual stresses of salt inhibition and organic toxicity. Sudden leaks can cause severe water and soil pollution, devastating regional ecosystems. High salinity can trigger plasmolysis in microorganisms, leading to the collapse of traditional activated sludge systems, soil compaction, and a surge in groundwater mineralization. Furthermore, toluene, due to its strong carcinogenicity and low water solubility, easily adsorbs and remains in sedimentary phases. Through biomagnification in the food chain, its carcinogenicity and genotoxicity pose a significant threat to higher organisms.

[0003] Currently, among methods for toluene removal, microbial degradation has advantages such as low cost and no secondary pollution. However, in high-salt environments, microbial degradation suffers from problems such as metabolic inactivation and a lack of functional microbial communities. Therefore, it is urgent to find a suitable microorganism for toluene degradation in high-salt environments and its degradation method. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a halophilic bacterium and its application. This strain is suitable for survival in high-salt environments containing toluene, and can remove toluene in high-salt environments.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] This invention provides a halophilic bacterium, strain name: Halophilic bacterium (Halophilic bacterium halophilum). Salinicola halophilus A3, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, on July 30, 2025, with accession number CGMCC No. 35460.

[0007] The aforementioned halophilic bacterium was obtained from activated sludge samples collected from the wastewater treatment plant of Qisheng Leather Factory in Hebei Province through enrichment culture and stepwise screening. When cultured for 24 h at 30℃ on solid LB medium containing 250 g / L NaCl, the colonies of this strain were regularly round, approximately 0.5 mm in diameter, white, smooth, raised, moist, opaque, and viscous.

[0008] The halophilic bacterium provided by this invention has high salt tolerance at room temperature and can be used to degrade toluene in high-salt environments, with a degradation rate of 85%-89% for toluene.

[0009] The present invention provides a microbial agent comprising the above-mentioned Halophilic halophyte and / or the fermentation broth of the above-mentioned Halophilic halophyte.

[0010] This invention does not impose any particular limitation on the dosage form of the microbial agent; it can be a solid dosage form, a liquid dosage form, or other types of dosage forms in the art. This invention can add excipients or other components to the microbial agent according to usage requirements.

[0011] The bacterial agent provided by this invention has high concentration salt resistance under normal temperature conditions and can be used to degrade toluene in high-salt environments, with a degradation rate of 85%-89% for toluene.

[0012] The present invention provides a fermentation method for the above-mentioned halophilic salt field strain, comprising the following steps: inoculating the above-mentioned halophilic salt field strain into a culture medium and fermenting it.

[0013] This invention provides the application of the above-mentioned halophilic bacterium in the degradation of toluene. The above-mentioned halophilic bacterium strain can be used to prepare products that degrade toluene.

[0014] This invention provides the application of the above-mentioned microbial agent in the degradation of toluene.

[0015] The present invention provides a method for preparing the above-mentioned microbial agent, comprising the following steps: inoculating the above-mentioned halophilic bacterium into a culture medium and fermenting it.

[0016] For example, Halophilic bacterium A3 can be inoculated into LB liquid medium and cultured in a shaker at 30°C and 120 r / min.

[0017] The present invention provides a method for degrading toluene, comprising the following steps: using the above-mentioned halophilic bacterium or the above-mentioned bacterial agent to degrade toluene.

[0018] Furthermore, the method includes the following steps: inoculating a substance containing toluene with halophilic bacterium or the above-mentioned bacterial agent, and then fermenting and culturing it.

[0019] Furthermore, the vaccination rate is 5%.

[0020] Furthermore, the culture temperature was 30℃.

[0021] Furthermore, the culture time is 3 days.

[0022] Using the Halophilic bacterium halophilum of the present invention to degrade toluene, under the conditions of salt concentration of 250 g / L and toluene concentration of 100 mg / L, the removal rate of toluene by Halophilic bacterium halophilum A3 can reach 85%-89%. Attached Figure Description

[0023] Figure 1 The results are for strain A3 cultured on a solid LB plate containing 250 g / L NaCl.

[0024] Figure 2 This is a phylogenetic tree.

[0025] Figure 3 This is a diagram showing the degradation effect of strain A3 on toluene in a high-salt environment. Detailed Implementation

[0026] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0027] The preparation method of LB liquid culture medium includes: 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract, mixed with water, pH 7.1-7.5, and sterilized at 120℃ for 30 min.

[0028] The preparation method of solid LB plates containing 250 g / L NaCl includes: 250 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract, and 20 g / L agar, mixed with water, pH 7.1-7.5, and sterilized at 120℃ for 30 min.

[0029] Preparation method of toluene-containing culture medium: Add toluene filtered through a 0.22 μm membrane to the sterilized culture medium.

[0030] Unless otherwise specified, all techniques or conditions used in the examples were performed using conventional methods or in accordance with techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents used, unless otherwise specified, were all conventional products that can be purchased from legitimate suppliers or prepared according to conventional methods in this field. Instruments used, unless otherwise specified, were all conventional instruments that can be purchased from legitimate suppliers.

[0031] Unless otherwise specified, all solutions of this invention are prepared using water as the solvent.

[0032] The following is a description through specific embodiments.

[0033] Example 1

[0034] The enrichment and screening of bacterial strains from activated sludge includes the following experimental methods:

[0035] (1) In November 2024, activated sludge was collected from the wastewater treatment plant of Qisheng Leather Factory in Hebei Province. 1.0 g of sludge sample was put into 100 mL of LB liquid medium and cultured in a shaker at 30℃ and 120 r / min for 3 days to enrich the strain and obtain the enriched culture solution.

[0036] (2) Take 10 mL of the enrichment culture medium from step (1) and place it in an enrichment bottle containing culture medium 1 (the volume of culture medium 1 is 100 mL). Incubate in a constant temperature shaking incubator at 30℃ and 120 r / min until the enrichment bottle becomes turbid.

[0037] Culture medium 1: Based on LB liquid culture medium, adjust the NaCl content to 50 g / L and add toluene to a final concentration of 20 mg / L.

[0038] (3) Take 10 mL of the enrichment culture medium from step (2) and place it in an enrichment bottle containing culture medium 2 (the volume of culture medium 2 is 100 mL). Incubate at 30℃ and 120 r / min until the enrichment bottle becomes turbid.

[0039] Culture medium 2: Based on LB liquid culture medium, adjust the NaCl content to 100 g / L and add toluene to a final concentration of 40 mg / L.

[0040] (4) Take 10 mL of the enrichment culture medium from step (3) and place it in an enrichment bottle containing culture medium 3 (the volume of culture medium 3 is 100 mL). Incubate at 30℃ and 120 r / min until the enrichment bottle becomes turbid.

[0041] Culture medium 3: Based on LB liquid culture medium, adjust the NaCl content to 150 g / L and add toluene to a final concentration of 60 mg / L.

[0042] (5) Take 10 mL of the enrichment culture medium from step (4) and place it in an enrichment bottle containing culture medium 4 (the volume of culture medium 4 is 100 mL). Incubate at 30℃ and 120 r / min until the enrichment bottle becomes turbid.

[0043] Culture medium 4: Based on LB liquid culture medium, adjust the NaCl content to 200 g / L and add toluene to a final concentration of 80 mg / L.

[0044] (6) Take 10 mL of the enrichment culture medium from step (5) and place it in an enrichment bottle containing culture medium 5 (the volume of culture medium 5 is 100 mL). Incubate at 30℃ and 120 r / min until the enrichment bottle becomes turbid.

[0045] Culture medium 5: Based on LB liquid culture medium, adjust the NaCl content to 250 g / L and add toluene to a final concentration of 100 mg / L.

[0046] (7) Take the enrichment culture medium from step (6) and perform 10 liters of sterile physiological saline. -1 -10 -9 Nine serial dilutions were performed, and 20 μL of each dilution was spread onto solid LB agar plates containing 250 g / L NaCl. The plates were inverted and incubated at 30°C for 3 days. Single white colonies were selected from the plates and streaked onto solid LB agar plates containing 250 g / L NaCl. This process was repeated three times to obtain pure bacteria, which were named A3 and preserved in glycerol.

[0047] Example 2

[0048] Strawberry strain A3 was streaked onto a solid LB agar plate containing 250 g / L NaCl and incubated at 30°C for 24 h. The morphological characteristics of the colonies were then observed. Figure 1 As shown, the colonies are regular circles, about 0.5 mm in diameter, white, with a smooth, raised, moist, opaque surface and a viscous texture.

[0049] The white strain A3 obtained from the screening was sent to Shanghai Meiji Biotechnology Co., Ltd., which performed 16S rRNA gene sequencing using primers 27F / 1492R.

[0050] Primer 27F: AGRGTTYGATYMTGGCTCAG (SEQ ID NO.1);

[0051] Primer 1492R: RGYTACCTTGTTACGACTT (SEQ ID NO.2);

[0052] All the primers mentioned above were provided by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0053] The 16S rRNA gene of strain A3 is shown in (SEQ ID NO.3).

[0054]

[0055] The sequencing results were compared using BLAST on the NCBI website, and a phylogenetic tree was constructed, such as... Figure 2 As shown, strain A3 was identified as belonging to Halophilic halophyte (Halophilus halophilus). Salinicola halophilus ).

[0056] Strain A3 was named Halophilic halophyte (Halophilic halophyte). Salinicola halophilus A3, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, on July 30, 2025, with accession number CGMCC No. 35460.

[0057] Example 3

[0058] The use of Halophilic bacterium A3 for toluene degradation in a high-salt environment includes the following steps:

[0059] (1) Inoculate the preserved halophilic bacterium A3 into LB liquid medium and culture it in a shaker at 30℃ and 120 r / min until the enrichment bottle becomes turbid, thus completing the activation of halophilic bacterium;

[0060] (2) Inoculate activated Halophilic bacterium A3 into a medium containing salt and toluene; the inoculation amount of Halophilic bacterium A3 is 5%, that is, the volume percentage of activated Halophilic bacterium A3 to the medium containing salt and toluene is 5%; the NaCl content in the medium containing salt and toluene is 250 g / L, and the toluene concentration is 100 mg / L; culture in a shaker at 30℃ and 120 r / min for 3 days to complete degradation.

[0061] Example 4

[0062] Referring to the method of Example 3, the concentrations of toluene in the culture media containing salt and toluene were set to 50 mg / L, 75 mg / L, 100 mg / L, 125 mg / L, and 150 mg / L, respectively, with the rest being the same as in Example 3.

[0063] The degradation rate of toluene was determined by gas chromatography, including the following steps: An AutoSystem XL gas chromatograph (PE Corporation, USA) equipped with an FID detector, a six-way quantitative valve, a packed column, and a PE-WAX standard capillary column (30 m × 0.53 mm × 1.0 μm). High-purity nitrogen was used as the carrier gas. A 1 mL airtight syringe was used to take a sample through the sampling port. The gas chromatography detection conditions were set as follows: vaporization chamber temperature 250℃, column temperature constant at 150℃, detector temperature 300℃, air flow rate 300 mL / min, hydrogen flow rate 15 mL / min, carrier gas (high-purity nitrogen) flow rate 1 mL / min, and split ratio set to 100:1.

[0064] The degradation rate of toluene was determined by gas chromatography. The degradation rate was calculated as follows: Degradation rate = (1 - Residual concentration / Original concentration) × 100%.

[0065] The degradation effect of halophilic bacterium A3 on toluene in high-salt environments is as follows: Figure 3 As shown, in a high-salt environment with a salt concentration of 250 g / L, when the toluene concentration in the culture medium was 100 mg / L, the Halophytum halophilum A3 exhibited the best degradation effect on toluene, with a degradation rate reaching 85.86%.

[0066] Repeating the above experiment multiple times, the removal rate of toluene by Halophilic bacterium A3 can reach 85%-89%.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A halophilic halophyte, characterized in that... It is a halophilic halophyte (Halophilic halophyte) Salinicola halophilus A3, with accession number CGMCC No.35460.

2. A microbial agent, characterized in that, Includes the halophilic halophyte bacterium described in claim 1.

3. The application of the halophilic bacterium described in claim 1 in the degradation of toluene.

4. The application of the microbial agent according to claim 2 in the degradation of toluene.

5. A fermentation method for the halophilic halophyte fungus according to claim 1, characterized in that, Includes the following steps: The halophilic halophyte bacterium described in claim 1 was inoculated into the culture medium and fermented.

6. A method for degrading toluene, characterized in that, The process includes the following steps: using the halophilic bacterium described in claim 1 or the bacterial agent described in claim 2 to degrade toluene.

7. The method for degrading toluene according to claim 6, characterized in that, The halophilic bacterium described in claim 1 or the bacterial agent described in claim 2 is inoculated into a substance containing toluene and fermented.

8. The method for degrading toluene according to claim 7, characterized in that, The vaccination rate is 5%.

9. The method for degrading toluene according to claim 7 or 8, characterized in that, The incubation temperature is 30℃.

10. The method for degrading toluene according to claim 7 or 8, characterized in that, The incubation period is 3 days.

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