A strain F19 and its application

Abstract

This invention discloses a strain F19, which is classified and named *Primatellis* F19. They are strain F19, deposited at the China Center for Type Culture Collection (CCTCC) on July 16, 2025, with accession number CCTCC NO: M 20251621, is disclosed in this invention. The invention also discloses its application in the degradation of benzophenone-3. Furthermore, this invention discloses the prepared bacterial agent and its application in the degradation of benzophenone-3. Strain F19 of this invention not only possesses highly efficient substrate degradation capabilities but also strong environmental adaptability, providing a highly efficient degrading strain resource for the biological reduction and control of residual benzophenone-3 in water bodies, soil, and other environments, and has significant theoretical and applied value.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a strain F19 and its applications. Background Technology

[0002] Benzophenone-3 (BP-3) is one of the most important members of the organic ultraviolet (UV) absorber family. Due to its excellent UV absorption properties, it can completely absorb UVB and some UVA radiation, and is therefore widely used in various personal care products such as sunscreens, cosmetics, and shampoos. Furthermore, BP-3 has good thermal stability, not decomposing at 200℃. Adding it to packaging materials, paints, and textiles can effectively prevent UV damage, avoiding cracking and fading. Therefore, compared to other benzophenone-based sunscreens, BP-3 is currently the most widely used UV absorber worldwide. In recent years, the unprecedented increase in public awareness of sun protection has led to the widespread use of sunscreen products in daily life, resulting in a large influx of its core ingredient, benzophenone-3, into the environment. Due to improper disposal or inadequate treatment technologies, benzophenone-3 is continuously released into aquatic and soil environments, becoming a new type of pollutant. Furthermore, because benzophenone-3 is a lipophilic molecule, it exhibits bioaccumulation. This means that once it enters an organism, benzophenone-3 will continuously accumulate and is difficult to eliminate through normal metabolism. Consequently, it not only has varying degrees of impact on the environment but also directly or indirectly harms human health. Therefore, strengthening the removal of residual benzophenone-3 from the environment is an issue that cannot be ignored and urgently needs to be addressed. Research on the degradation of benzophenone-3 pollutants has become another key focus in the field of trace pollutant research.

[0003] Currently, the main methods for remediating benzophenone-3 include physical, chemical, and biological methods. Physical methods primarily employ membrane treatment, which offers advantages such as high removal rates, no secondary pollution, and simple operation, but suffers from drawbacks such as high cost and limited membrane lifespan. Chemical methods, while highly efficient, also have disadvantages such as high cost and susceptibility to secondary pollution. Biological methods, which eliminate pollutants through microbial metabolism, offer advantages such as high efficiency, economy, safety, and no secondary pollution, playing a crucial role in the degradation and remediation of benzophenone residues. Therefore, screening for efficient, stable, and environmentally adaptable benzophenone-3 degrading strains will provide microbial strain resources and technical support for addressing benzophenone pollution in the environment. Summary of the Invention

[0004] The purpose of this invention is to provide a strain F19 and its applications to address the shortcomings of existing technologies.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides a strain F19, which is classified and named as *Primatellis* F19. Priestia sp. F19, deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC NO: M 20251621.

[0007] A second aspect of the present invention provides the application of the above-mentioned strain F19 in the degradation of benzophenone-3.

[0008] Furthermore, the degradation of benzophenone-3 includes the degradation of benzophenone-3 in water bodies and soil.

[0009] The third aspect of the present invention provides the application of the above-mentioned strain F19 in the preparation of a bacterial agent that degrades benzophenone-3.

[0010] A fourth aspect of the present invention provides a bacterial agent prepared from the above-mentioned strain F19.

[0011] Furthermore, the microbial agent is prepared by the following steps:

[0012] (1) Inoculate strain F19 in test tubes into the first liquid culture medium and culture it at 25~37℃ and 180~220 rpm until the logarithmic phase, which is then used to inoculate the seed liquid in the fermenter;

[0013] (2) Add the second liquid culture medium to the fermenter, then sterilize by high temperature and moist heat. After cooling to 30~32℃, inoculate the seed liquid obtained in step (1) into the fermenter at an inoculation rate of 5~8 v / v% for fermentation. Fermentation conditions: fermentation temperature is 30~32℃, sterile air flow rate is 1.0~1.5 vvm, stirring speed is 280~350 rpm, and fermentation time is 24~48h. After fermentation, the number of cells in the fermentation liquid reaches 10 9 CFU / mL or higher;

[0014] (3) After fermentation, the fermentation liquid is directly packaged into liquid dosage form in plastic packaging buckets or bottles.

[0015] Furthermore, in step (1), the first liquid culture medium includes LB liquid culture medium.

[0016] Furthermore, in step (2), the second liquid culture medium is formulated as follows: glucose 1.0 w / v%, peptone 0.5 w / v%, yeast extract 0.4 w / v%, (NH4)2SO4 0.2 w / v%, K2HPO4 0.1 w / v%, MgSO4•7H2O 0.02 w / v%, NaCl 0.5 w / v%, CaCl2 0.2 w / v%, natural pH.

[0017] The fifth aspect of the present invention provides the application of the above-mentioned microbial agent in the degradation of benzophenone-3.

[0018] Furthermore, the degradation of benzophenone-3 includes the degradation of benzophenone-3 in water bodies and soil.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention isolates a strain F19 from activated sludge samples that can efficiently degrade benzophenone-3, completely removing 0.1 mM benzophenone-3 in approximately 9 hours. Biologically, strain F19 is related to... Priestia megaterium NBRC 15308 T It has the highest homology. Priestia Currently, there are no reports of benzophenone-3 degradation capabilities, but this invention has made a novel discovery. Priestia The benzophenone-3 degradation ability of sp. F19 provides an important degrading strain resource for the biological reduction and control of benzophenone-3 residues in water, soil and other environments, and has excellent degradation ability and good application prospects.

[0021] 2. The strain F19 screened in this invention can effectively degrade benzophenone-3 in a temperature range of 25~37℃ and a pH range of 5~9, indicating that strain F19 has excellent environmental adaptability and good application potential.

[0022] 3. This invention utilizes strain F19 to degrade benzophenone-3. Compared with physical and chemical methods, the cost is significantly reduced, and there is no accumulation of toxic byproducts. It is of great significance for the bio-enhanced remediation of benzophenone-3 in water bodies, soil and other environments.

[0023] In summary, the strain F19 screened in this invention can be applied to the field of bioremediation of benzophenone-3 residues in water, soil and other environments. This strain not only has a high efficiency in substrate degradation but also a strong environmental adaptability, providing a highly efficient degradation strain resource for the bioreduction and control of benzophenone-3 residues in water, soil and other environments, and has important theoretical and applied value. Attached Figure Description

[0024] Figure 1The HPLC chromatogram shows the degradation of benzophenone-3 by strain F1.

[0025] Figure 2 Photograph (A) and scanning electron microscope image (B) of the colony morphology of strain F19 on LB solid medium.

[0026] Figure 3 Phylogenetic tree of the 16S rRNA gene of strain F19.

[0027] Figure 4 The degradation characteristics of strain F19 under YMS liquid medium supplemented with different concentrations of yeast extract.

[0028] Figure 5 The degradation characteristics of strain F19 under different initial inoculum amounts.

[0029] Figure 6 The degradation characteristics of strain F19 under different pH conditions.

[0030] Figure 7 The degradation characteristics of strain F19 under different temperature conditions.

[0031] Information on the preservation of biological materials

[0032] F19, classified and named as *Primatellis* genus F19. Priestia sp. F19, Latin name Priestia sp., deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 16, 2025, with accession number CCTCC NO: M 20251621. Detailed Implementation

[0033] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] The culture medium formulations involved in the following examples are as follows:

[0035] The formula for inorganic salt liquid culture medium (hereinafter referred to as MSM) is as follows (1 L): NaCl 1.0 g, NH4Cl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, and ultrapure water to a final volume of 1 L. Sterilize at 121℃ for 20 min. Natural pH.

[0036] The formulation of 1 / 4R2A solid culture medium (1 L) is as follows: 0.8 g of R2A (Haibo Biotechnology Co., Ltd., product number: HB0167-2), ultrapure water to a final volume of 1 L, 18 g of agar powder, sterilized at 121℃ for 20 min. Natural pH.

[0037] The YMS liquid culture medium formula (1 L) is as follows: NaCl 1.0 g, NH4Cl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, yeast extract 2.0 g, and ultrapure water to a final volume of 1 L. Sterilize at 121℃ for 20 min. Natural pH.

[0038] The LB liquid culture medium formula (1 L) is as follows: NaCl 5.0 g, yeast extract 5.0 g, peptone 10.0 g, ultrapure water to a final volume of 1 L, sterilized at 121℃ for 20 min. Natural pH.

[0039] The LB solid medium formula (1 L) is as follows: NaCl 5.0 g, yeast extract 5.0 g, peptone 10.0 g, ultrapure water to a final volume of 1 L, agar powder 18 g, sterilized at 121℃ for 20 min. Natural pH.

[0040] Unless otherwise specified, the petri dishes (plates) involved in the following examples have a diameter of 90 mm.

[0041] The following examples involve adding benzophenone-3 by adding benzophenone-3 stock solution to the appropriate concentration, wherein the concentration of benzophenone-3 stock solution is 20 mM, and DMSO is used as the solvent.

[0042] Example 1 Isolation of strain F19

[0043] 5.0 g of activated sludge sample from Nanjing Baorui Renewable Resources Green Sorting Center in Jiangsu Province was added to 100 mL of MSM containing a final concentration of 0.2 mM benzophenone-3 (BP-3). The sample was cultured at 30℃ and 180 rpm for 7 days. The sample was then transferred to fresh MSM containing a final concentration of 0.2 mM benzophenone-3 at a 5% inoculum (v / v). The enrichment culture was repeated three times. The concentration of benzophenone-3 in the fourth-generation enrichment solution was detected by high performance liquid chromatography (HPLC). The enrichment solution with benzophenone-3 degradation effect was diluted and spread onto the surface of 1 / 4 R2A solid medium containing a final concentration of 0.2 mM benzophenone-3. The culture was incubated at 30℃ for 2–4 days. Single colonies were picked and transferred to test tubes containing 3 mL LB liquid medium, and incubated at 30℃ and 180 rpm until the logarithmic phase. The obtained bacterial culture was then inoculated at a 5% inoculum (v / v) into MSM containing a final concentration of 0.2 mM benzophenone-3, and incubated at 30℃ and 180 rpm for 3 days. 500 μL of the culture sample was taken, and an equal volume of acetonitrile was added. After thorough shaking and mixing, the sample was centrifuged at 12000 rpm for 1 min. The supernatant sample was filtered through a 0.22 μm organic filter membrane, and the concentration of benzophenone-3 in the sample was detected by HPLC. If the concentration of benzophenone-3 decreased, it was inferred that the strain had degradation ability.

[0044] Chromatographic conditions for HPLC determination of benzophenone-3 concentration: Agilent ZORBAX SB C18 column (250 mm × 4.6 mm × 5 μm); mobile phase: acetonitrile: 0.1 v / v% formic acid aqueous solution (v:v = 70:30); flow rate: 1.0 mL / min. -1 The detection wavelength was 290 nm; the column temperature was 35°C; and the injection volume was 40 μL. The same applies below.

[0045] A benzophenone-3 degrading strain was successfully screened using enrichment culture and dilution plating isolation method, and named F19.

[0046] Strain strain F19 was inoculated into 100 mL of LB liquid medium and incubated at 30°C and 180 rpm. Fermentation broth was obtained after culturing for 12 h. The fermentation broth was centrifuged at 6000 rpm for 5 min, the supernatant was removed, and the bacterial cells were collected as a precipitate. The bacterial cells were washed twice with MSM, and finally resuspended in 10 mL of MSM as seed culture for strain F19.

[0047] The prepared seed culture of strain F19 was inoculated into 20 mL of YMS liquid medium containing 0.1 mM benzophenone-3 to allow it to reach its final OD. 600The concentration was 1.0. Samples were taken after culturing at 30℃ and 180 rpm for 3 days. A control group was set up with uninoculated YMS liquid medium containing a final concentration of 0.1 mM benzophenone-3. The culture conditions were the same, and samples were taken after 3 days of culture. The concentration of residual benzophenone-3 was determined by HPLC, and the results are as follows: Figure 1 As shown, this indicates that F19 can degrade benzophenone-3.

[0048] Example 2 Identification of strain F19

[0049] Strain F19 was cultured on LB solid medium at 30℃ for 1-2 days. The colonies were round, pale yellow, with neat edges and smooth surfaces. Figure 2 A). The main physiological and biochemical characteristics of strain F19 are that it is a Gram-positive bacterium, and the cell morphology is rod-shaped, approximately 2.3 μm long and 0.45 μm wide. Figure 2 B), obligate aerobic; VP reaction shows red, indicating a positive result; indole reaction is negative; methyl red reaction shows yellow, indicating a negative result. Strain F19 is resistant to ampicillin. Using fresh bacterial culture of strain F19 as a template, 16S rRNA gene sequence universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO.1) and 1492R (5'-TACGGCTACCTTGTTACGACTT-3', as shown in SEQ ID NO.2) were used for amplification. The amplified product was purified and ligated into the vector pMD19-T. Sequencing yielded a gene sequence of 1474 bp (as shown in SEQ ID NO.3). This sequence was compared and analyzed in the EzBioCloud database (https: / / www.ezbiocloud.net), and a phylogenetic tree was constructed. The results showed that strain F19 is resistant to ampicillin. Priestia megaterium NBRC 15308 T The highest homology ( Figure 3 The homology reaches 100%.

[0050] Based on the colony morphology, physiological and biochemical characteristics, and 16S rRNA gene phylogenetic tree of strain F19, it was identified as belonging to the genus *Primate*. Priestia (sp.), has been deposited at the China Center for Type Culture Collection, accession number CCTCCNO: M 20251621.

[0051] Example 3 Degradation characteristics test of strain F19

[0052] 3.1 Preparation of seed culture for strain F19

[0053] The seed culture of strain F19 used in the following experiments was prepared in the same manner as in Example 1.

[0054] 3.2 Degradation characteristics of strain F19 in YMS liquid medium supplemented with different concentrations of yeast extract

[0055] The prepared seed culture of strain F19 was inoculated into 20 mL of YMS liquid medium containing 0 g / L yeast extract (MSM), 0.2 g / L yeast extract (0.2 g / L YMS), 0.5 g / L yeast extract (0.5 g / L YMS), 1 g / L yeast extract (1 g / L YMS), and 2 g / L yeast extract (2 g / L YMS) respectively, to allow its final OD to reach 0.1 mM. 600 All samples were 0.5 μL and cultured at 30℃ and 180 rpm for 24 h, with samples taken periodically during the culture process: every 3 h for the first 12 h, and every 12 h thereafter. YMS liquid medium containing different concentrations of yeast extract from uninoculated culture with a final concentration of 0.1 mM benzophenone-3 was set as a blank control group (CK), with experimental conditions consistent with each treatment group. Each blank control group and each treatment group was replicated in triplicate. The concentration of residual benzophenone-3 was determined by HPLC. Results are as follows: Figure 4 As shown ( Figure 4 Only one control group (CK) is shown in the table. In YMS liquid medium with different concentrations of yeast extract, strain F19 showed the highest degradation rate of benzophenone-3 in 0.5 g / L YMS, while in MSM without yeast extract, strain F19 could hardly degrade benzophenone-3, indicating that strain F19 requires an additional carbon source to provide energy during the degradation of benzophenone-3.

[0056] 3.3 Degradation characteristics of strain F19 under different initial inoculum amounts

[0057] The prepared seed culture of strain F19 was added to 20 mL of YMS liquid medium containing 0.5 g / L yeast extract, and the OD was adjusted. 600 The concentrations of benzophenone-3 stock solution were added to a final concentration of 0.1 mM at concentrations of 0.1, 0.3, 0.5, 0.7, and 1.0, respectively. The cultures were incubated at 30°C and 180 rpm for 24 h, with samples taken periodically during the incubation process: every 4 h for the first 12 h, and every 12 h thereafter. A blank control group (CK) was set up using YMS liquid medium containing 0.5 g / L yeast extract from uninoculated culture at a final concentration of 0.1 mM benzophenone-3. The experimental conditions were the same as for each treatment group. Three replicates were set up for both the blank control group and each treatment group. The residual benzophenone-3 concentration was determined by HPLC. The results are as follows: Figure 5 As shown, OD 600 Between 0.1 and 1.0, the higher the inoculum size of strain F19, the faster the degradation rate of benzophenone-3.

[0058] 3.4 Degradation characteristics of strain F19 under different pH conditions

[0059] The prepared seed culture of strain F19 was added to 20 mL of YMS liquid medium containing 0.5 g / L yeast extract at different pH values ​​(adjusted to 5, 6, 7, 8, and 9 with 5 M NaOH and 5 M HCl, respectively), and the OD was adjusted. 600 The concentration of benzophenone-3 stock solution was increased to 0.1 mM, and the culture was carried out at 30℃ and 180 rpm for 12 h, with samples taken every 3 h during the culture process. YMS liquid medium containing 0.5 g / L yeast extract at different pH values ​​(pH 5, 6, 7, 8, and 9) of the uninoculated bacterial culture with a final concentration of 0.1 mM benzophenone-3 served as a blank control group (CK). The experimental conditions were the same as those for each treatment group. Each blank control group and each treatment group was replicated in triplicate. The concentration of residual benzophenone-3 was determined by HPLC. The results are as follows: Figure 6 As shown ( Figure 6 Only one group (CK) was presented. Under pH conditions of 5-9, strain F19 could degrade benzophenone-3 relatively quickly and completely remove 0.1 mM benzophenone-3 within 12 hours. The optimal degradation pH was 9, in which 0.1 mM benzophenone-3 could be completely removed in about 9 hours.

[0060] 3.5 Degradation characteristics of strain F19 under different temperature conditions

[0061] The prepared seed culture of strain F19 was added to 20 mL of YMS liquid medium containing 0.5 g / L yeast extract, and the OD was adjusted. 600The initial concentration of benzophenone-3 was 0.5, and benzophenone-3 stock solution was added to a final concentration of 0.1 mM. Samples were collected after culturing at different temperatures (16℃, 25℃, 30℃, 37℃, and 42℃) and 180 rpm for 8 h. A blank control group (CK) was set up using YMS liquid medium containing 0.5 g / L yeast extract from uninoculated culture with a final concentration of 0.1 mM benzophenone-3. The experimental conditions were the same as for each treatment group, i.e., culturing at different temperatures (16℃, 25℃, 30℃, 37℃, and 42℃) and 180 rpm for 8 h. Three replicates were set up for each blank control group and each treatment group. The residual benzophenone-3 concentration was detected by HPLC, and the degradation rate was calculated using the formula: Degradation rate (%) = (Initial concentration - Residual concentration) / Initial concentration × 100%, where the initial concentration is the initial concentration of benzophenone-3, and the residual concentration is the remaining concentration of benzophenone-3. The results are as follows: Figure 7 As shown ( Figure 7 Only one CK group was presented in the study. At 25~37℃, strain F19 showed a high degradation rate of benzophenone-3, with the optimal degradation temperature being 30℃. When the temperature was higher than 37℃ or lower than 25℃, the degradation rate of benzophenone-3 by strain F19 decreased significantly.

[0062] Example 4: Preparation of F19 bacterial agent

[0063] Strain F19 was activated on LB solid medium slant in test tubes at 30°C, and its degradation performance on benzophenone-3 was measured to ensure its functionality. Colonies grown on the slant (test tube inoculum) were inoculated into four 1000 mL Erlenmeyer flasks containing 400 mL LB liquid medium and cultured at 30°C and 180 rpm until the logarithmic growth phase. These colonies were then used to inoculate the seed culture in the fermenter. The fermenter was 50 L, with a feed volume of 30 L. The culture medium formulation (m / v%) was: glucose 1.0%, peptone 0.5%, yeast extract 0.4%, (NH4)2SO4 0.2%, K2HPO4 0.1%, MgSO4•7H2O 0.02%, NaCl 0.5%, CaCl2 0.2%, natural pH. After feeding, the cultured seed culture was sterilized at 115℃ for 30 min using a high-temperature moist heat method. After cooling to 30℃, the cultured seed culture was inoculated into the fermenter at an inoculation rate of 5 v / v%. During the inoculation process, the fermenter temperature was maintained at 30℃, the sterile air flow rate was 1.0 vvm (i.e., volume air (L) / volume fermentation broth (L) / min), the stirring speed was 300 rpm, and the fermentation time was 48 h. After fermentation, the cell count in the fermentation broth reached 10⁻⁶. 9 CFU / mL or higher.

[0064] After fermentation, the fermentation liquid is removed from the tank and directly packaged into liquid dosage forms using plastic packaging buckets or bottles.

Claims

1. A bacterial strain F19, characterized in that, The classification name is Pseudomonas sp. Priestia , preserved in the China Center for Type Culture Collection on July 16, 2025, with the preservation number CCTCC NO: M20251621.

2. The application of strain F19 according to claim 1 in the degradation of benzophenone-3, wherein the application temperature is 25~37℃ and the pH is 5~9.

3. Use according to claim 2, characterized in that, The degradation of benzophenone-3 refers to the degradation of benzophenone-3 in water and soil.

4. The application of strain F19 according to claim 1 in the preparation of a bacterial agent for degrading benzophenone-3, wherein the application temperature is 25~37℃ and the pH is 5~9.

5. A bacterial agent prepared using the strain F19 of claim 1.

6. The bacterial agent of claim 5, characterized in that, The microbial agent is prepared by the following steps: (1) Inoculate strain F19 in test tubes into the first liquid culture medium and culture it at 25~37℃ and 180~220 rpm until the logarithmic phase, which is then used to inoculate the seed liquid in the fermenter; (2) Put the second liquid culture medium into the fermenter, then high-temperature and high-humidity sterilize, cool to 30-32℃, then inoculate the seed liquid obtained in step (1) into the fermenter at an inoculation amount of 5-8 v / v% to carry out fermentation, the fermentation conditions are: the fermentation temperature is 30-32℃, the aeration amount of sterile air is 1.0-1.5 vvm, the stirring speed is 280-350 rpm, and the fermentation time is 24-48 h; after the fermentation is completed, the number of bacteria in the fermentation liquor reaches more than 10 9 CFU / mL. (3) After fermentation, the fermentation liquid is directly packaged into liquid dosage form in plastic packaging buckets or bottles.

7. The bacterial agent of claim 6, wherein In step (1), the first liquid culture medium includes LB liquid culture medium.

8. The bacterial agent of claim 6, wherein In step (2), the second liquid culture medium is formulated as follows: glucose 1.0 w / v%, peptone 0.5 w / v%, yeast extract 0.4 w / v%, (NH4)2SO4 0.2 w / v%, K2HPO4 0.1 w / v%, MgSO4•7H2O 0.02 w / v%, NaCl 0.5 w / v%, CaCl2 0.2 w / v%, natural pH.

9. The application of the microbial agent according to any one of claims 5-8 in the degradation of benzophenone-3, wherein the application temperature is 25-37°C and the pH is 5-9.

10. Use according to claim 9, characterized in that, The degradation of benzophenone-3 refers to the degradation of benzophenone-3 in water and soil.

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