Pseudomonas gilardiia with high efficiency of degrading phenols and pesticides and application thereof

Abstract

The application discloses a Pseudomonas gilardiis capable of efficiently degrading phenols and pesticides and an application thereof, and belongs to the technical field of environmental microorganisms and biological remediation. The Pseudomonas gilardiis has a preservation number of CGMCC NO. 37396 and a preservation date of January 15, 2026. The Pseudomonas gilardiis obtained by separation has a broad-spectrum and efficient degradation capability for various phenolic compounds and organic pesticides. The strain breaks the limitation of single function of most degrading bacteria, and can simultaneously and efficiently treat two types of pollutants, i.e., phenols and pesticides.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and bioremediation technology, specifically involving a novel strain of *Pseudomonas galvanus* that can efficiently degrade various phenolic compounds and organic pesticides, and its application in the preparation of bioremediation agents for environmental pollution. Background Technology

[0002] Phenolic compounds (such as phenol and chlorophenol) and organopesticides (such as chlorpyrifos and carbofuran) are typical persistent organic pollutants that are widely present in the environment. They originate from industrial wastewater from chemical, pesticide, and pharmaceutical industries, as well as agricultural runoff. They are highly toxic, difficult to degrade, and bioaccumulate, posing a serious threat to aquatic ecosystems and human health.

[0003] Currently, methods for treating such pollutants mainly include physical adsorption, chemical oxidation, and advanced oxidation technologies. While these methods are effective, they generally suffer from drawbacks such as high cost, complex operation, and the potential for secondary pollution. In contrast, microbial degradation technology shows great potential due to its environmental friendliness, low cost, and ability to completely mineralize pollutants into CO2 and H2O.

[0004] Pseudomonas are common degrading bacteria in the environment, but different strains exhibit significant differences in their degradation spectrum and efficiency. Existing strains often only degrade a specific type of pollutant; for example, some strains are specific to degrading phenol but have weak degradation abilities for chlorophenols or pesticides. In real-world polluted environments, pollutants are usually diverse and complex systems; therefore, there is an urgent need to discover broad-spectrum degrading strains capable of simultaneously and efficiently degrading multiple different types of pollutants.

[0005] As a relatively little-studied species, *Pseudomonas gerzenetus*'s potential for broad-spectrum degradation of complex organic pollutants has not yet been fully explored. Therefore, isolating and obtaining a new strain of *Pseudomonas gerzenetus* with efficient and broad-spectrum degradation capabilities is of great significance for developing efficient environmental bioremediation technologies. However, *Pseudomonas gerzenetus* strains capable of simultaneously and efficiently degrading phenolic compounds and organopesticides have not yet been reported. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to overcome the shortcomings of the prior art and provide a new strain of Pseudomonas galvaniculatus with a broad degradation spectrum and high degradation efficiency.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The first aspect of the present invention provides a strain of *Pseudomonas galbana* (…). Pseudomonas guezennei (), its accession number is CGMCC NO.37396.

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

[0010] A second aspect of the present invention provides the fermentation broth or bacterial suspension of the aforementioned Pseudomonas galenae.

[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 may also be 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] A third aspect of the present invention proposes the use of the above-mentioned Pseudomonas galenae, its fermentation broth or bacterial suspension in the degradation of phenolic compounds and / or organic pesticides.

[0014] Preferably, the phenolic compounds include, but are not limited to, one or more of phenol, cresol, aminophenol, nitrophenol, naphthol, and chlorophenol.

[0015] Preferably, the organic pesticides include, but are not limited to, any one or more of organophosphates, organochlorines, pyrethroids, and carbamates. Specifically, the organophosphates include chlorpyrifos, trichlorfon, dichlorvos, dimethoate, malathion, and phoxim.

[0016] A fourth aspect of the present invention proposes the use of the above-mentioned Pseudomonas galenae, its fermentation broth or bacterial suspension in the preparation of microbial preparations that degrade phenolic compounds and / or organic pesticides.

[0017] The fifth aspect of the present invention provides a microbial preparation for degrading phenolic compounds and / or organic pesticides, the main components of which include one or more of the above-mentioned Pseudomonas galvanus, its fermentation broth or bacterial suspension.

[0018] Preferably, the microbial preparation further includes a pharmaceutically acceptable carrier.

[0019] "Pharmaceutical acceptable" means non-toxic materials that do not reduce the active ingredient. Such pharmaceutically acceptable excipients and carriers are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Company (1990) and hand book of Pharmaceutical Excipients, 3rd edition, edited by A. Kibbe, Pharmaceutical Press (2000)).

[0020] Preferably, the carrier includes, but is not limited to, one or more of the following: diluent, adhesive, wetting agent, disintegrant, absorption promoter, surfactant, and lubricant.

[0021] Preferably, the microbial preparation is formulated into a pharmaceutically acceptable dosage form.

[0022] Preferably, the dosage form includes tablets, capsules, granules, powders, granules, drops, emulsions, or suspensions.

[0023] The sixth aspect of the present invention also proposes the application of the above-mentioned microbial preparations in the bioremediation of environmental pollution.

[0024] This invention also proposes a method for degrading phenolic compounds and / or organic pesticides, comprising the following steps: adding the aforementioned *Pseudomonas galbana* to the phenolic compounds and / or organic pesticides. Pseudomonas guezennei ( ), its fermentation broth, bacterial suspension, or any one or more of them.

[0025] The beneficial effects of this invention are as follows: 1. The *Pseudomonas galenae* isolated by this invention exhibits a broad-spectrum and highly efficient degradation ability for a variety of phenolic compounds and organic pesticides.

[0026] 2. This strain breaks through the limitation of most degrading bacteria having a single function; a single strain can simultaneously and efficiently treat two major categories of significantly different pollutants: phenols and pesticides.

[0027] 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

[0028] Figure 1 This is a photograph of the *Pseudomonas geyseris* strain used in Example 1 of this invention. Figure 2 This is the NJ phylogenetic tree diagram in Embodiment 2 of the present invention; Figure 3 This is a graph showing the degradation time of phenol concentration by the strain in Example 4 of the present invention; Figure 4 This is a time-varying curve of the phenol degradation rate of the strain in Example 4 of the present invention; Figure 5 This is a graph showing the degradation time of chlorpyrifos concentration by the strain in Example 5 of the present invention; Figure 6 This is a time-varying curve of the degradation rate of chlorpyrifos by the strain in Example 5 of the present invention. Detailed Implementation

[0029] 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.

[0030] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0031] 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.

[0032] The culture media involved in the following examples are as follows: NB liquid culture medium: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, 1000 mL distilled water.

[0033] NB solid culture medium: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, 1000 mL distilled water, 20.0 g agar.

[0034] pH adjustment of NB liquid medium and NB solid medium: Adjust the pH of NB liquid medium or NB solid medium to neutral as needed, using 5 mol / L sodium hydroxide solution and 0.1 mol / L hydrochloric acid solution.

[0035] The present invention will be further described below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0036] Example 1: Separation and Purification Methods The isolation and purification method for *Pseudomonas geyseris* strains is as follows: S1: Take 100 μL of sample, which is fermentation broth produced from the natural fermentation of shrimp shells; in a clean bench, add 900 μL of sterile water to the sample, shake for 1 minute to evenly disperse the sample in the diluent to form a suspension; after dispersion, pipette 100 μL of the suspension into 900 μL of sterile water to obtain a 10-fold dilution, and then dilute it 10 times in sequence to obtain 10... 2 10 times dilution solution 3 10 times dilution solution 4 10 times dilution solution 510 times dilution solution 6 10 times dilution solution 7 10 times dilution and 10 8 The solution was diluted multiple times, and the entire process was carried out in a clean bench.

[0037] S2: Take 100ul of 10 3 10 times dilution solution 4 10 times dilution solution 5 10 times dilution solution 6 10 times dilution solution 7 10 times dilution and 10 8 The diluted solution was spread onto agar solid culture plates, and then the culture plates were placed in an incubator at 36.5℃ for 2-3 days until bacterial plaques appeared on the culture plates.

[0038] S3: After the culture is completed, according to the colony growth, single plaques of different shapes, colors and sizes are picked from the appropriate dilution gradient plates and streaked to isolate the *Pseudomonas galbana* as described in this invention.

[0039] The *Pseudomonas galbana* strain of this invention has short rod-shaped colonies with a round shape and neat edges. (e.g.) Figure 1 (As shown) S4: The purified bacterial strain was picked and inoculated into a 250ml Erlenmeyer flask containing 150ml of liquid culture medium. The flask was cultured at 36.5℃ and 200rpm for 13 hours to obtain a seed culture. The seed culture was then mixed with glycerol at a volume ratio of 7:3 to obtain a concentration of 1.0×10⁻⁶. 8 ~2.8×10 9 After marking the glycerol seed culture at 1000 / ml on the tube wall, store at -20℃ or for long-term storage at -80℃.

[0040] Example 2: PCR amplification and sequencing of 16S rDNA sequence S1: Genomic DNA was extracted using the Omega Bacterial DNA Kit (D3350-01). First, take 2 ml of the glycerol seed culture described in Example 1 into a sterile 2 ml centrifuge tube, centrifuge at 12000 rpm for 2 min, discard the supernatant and retain the precipitate; then, add 100 μl of 1×TE Buffer to the precipitate, vortex to mix, add 10 μl of lysozyme and mix, incubate at 37°C for 10 min; add 100 μl of BTL Buffer and 20 μl of proteinase K, mix, incubate at 55°C for 1 h, vortexing three times during incubation; add 5 μl of RNase A enzyme, mix, let stand at room temperature for 5 min, centrifuge at 10000 rpm for 2 min, and transfer 200 μl of supernatant to a new sterile 1.5 ml centrifuge tube; add 200 μl of BTL Buffer, mix, and incubate at 65°C for 10 min; add 200 μl of anhydrous ethanol, vortex to mix, transfer the entire sample to the adsorption column, centrifuge at 10000 rpm for 2 min, discard the supernatant and the adsorption column, and place the adsorption column into a new collection tube; add 500 μl of HBC to the adsorption column. Centrifuge at 10,000 rpm for 2 min and discard the supernatant. Add 700 μL DNA Wash Buffer to the adsorption column, centrifuge at 10,000 rpm for 2 min and discard the supernatant. Repeat twice. Place the empty adsorption column back into the collection tube and centrifuge at 10,000 rpm for 2 min. Add 30 μL to 50 μL of Elution Buffer (preheated to 65 °C) to the adsorption column to dissolve the DNA precipitate, thus obtaining genomic DNA. Store at -20 °C for later use.

[0041] S2: PCR amplification of 16S rDNA sequence. Using the genomic DNA obtained in step S1 as a template, Eubac27F and Eubac1492R were used as primers for PCR amplification. The PCR reaction system (50 μl) is as follows: The PCR reaction procedure was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, and repeated the denaturation, annealing and extension processes 30 times; extension at 72℃ for another 10 min, and storage of PCR amplification products at 4℃.

[0042] The sequence of the upstream primer Eubac27F is: agagtttgat cctggctcag (SEQ ID NO.2). The sequence of the downstream primer Eubac1492R is: ggttaccttg ttacgactt (SEQ ID NO.3). S3: Nucleic acid electrophoresis of PCR products. Take 5 μL of the PCR product obtained in step S2 and perform nucleic acid electrophoresis at 120V for 25 min. The 16S rDNA fragment amplified using the genomic DNA of the Pseudomonas strain described in this invention as a template has a single and high-brightness band.

[0043] S4: Sequencing of the 16S rDNA sequence. After purification and recovery of the PCR product obtained in step S2, 30 μL of the purified product was sent to Anhui Chuzhou General Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results showed that the 16S rDNA sequence of *Pseudomonas galvanis* of this invention is 1404 bp in length, and the specific sequence is shown below: SEQ ID NO.1 Example 3: Constructing a phylogenetic tree The 16S rDNA sequence of the *Pseudomonas geyseris* strain described in this invention was input into NCBI for BLAST alignment, and an NJ phylogenetic tree was constructed based on the barcode 16S rDNA segment sequence (e.g., ...). Figure 2 As shown), according to the phylogenetic tree, the strain obtained in this invention is highly similar to most sequences in the 16S rDNA barcode database of *Pseudomonas gerzenae*, but there is no complete overlap, nor is there a unique neighboring species sequence. Therefore, it can be concluded that the strain obtained in this invention belongs to *Pseudomonas gerzenae* in taxonomy. Pseudomonas guezennei This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 15, 2026, with accession number CGMCC NO.37396, and named AHMU-GZN-10111.

[0044] Example 4: Degradation of phenol by the strain Prepare an inorganic salt culture medium with 500 mg / L phenol as the sole carbon source, and inoculate it with 5% (v / v) of DEG-21 bacterial suspension in the logarithmic growth phase (OD200). 600 (≈1.0), and cultured at 30°C and 180 rpm with shaking. Samples were taken at regular intervals, and the residual phenol concentration was determined using the 4-aminoantipyrine spectrophotometric method. The results showed that after 24 hours of culture, the degradation rate of phenol reached 90-95%, while the concentration in the uninoculated control group showed no significant change. (e.g., ≈1.0) Figures 3-4 (As shown) Example 5: Degradation of chlorpyrifos by strain DEG-21 An inorganic salt culture medium with 50 mg / L chlorpyrifos as the sole carbon source was prepared, and the inoculation method was the same as in Example 1. The medium was incubated at 30°C with shaking at 180 rpm. The residual concentration of chlorpyrifos was detected by gas chromatography. The results showed that after 48 hours of incubation, the degradation rate of chlorpyrifos reached 90-95%. Figures 5-6 (As shown) 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 *Pseudomonas galbana*, characterized in that, Its accession number is CGMCC NO.37396.

2. The *Pseudomonas galbana* according to claim 1, characterized in that, The 16S rDNA sequence of this strain is shown in SEQ ID NO.

1.

3. The fermentation broth or bacterial suspension of *Pseudomonas galbana* as described in claim 1.

4. The use of *Pseudomonas galenae* as described in claim 1, the fermentation broth or bacterial suspension as described in claim 3, in the degradation of phenolic compounds and / or organic pesticides.

5. The application according to claim 4, characterized in that, The phenolic compound is one or more of phenol, cresol, aminophenol, nitrophenol, naphthol, and chlorophenol.

6. The application according to claim 4, characterized in that, The organic pesticide is any one or more of organophosphates, organochlorines, pyrethroids, and carbamates.

7. The use of *Pseudomonas galenae* as described in claim 1, the fermentation broth or bacterial suspension as described in claim 3, in the preparation of microbial preparations for degrading phenolic compounds and / or organic pesticides.

8. A microbial preparation for degrading phenolic compounds and / or organic pesticides, characterized in that, Its main components include one or more of the following: Pseudomonas galenae as described in claim 1, fermentation broth or bacterial suspension as described in claim 3.

9. The microbial preparation according to claim 8, characterized in that, The microbial preparation also includes a pharmaceutically acceptable carrier.

10. The microbial preparation according to claim 8, characterized in that, The microbial preparation is formulated into a pharmaceutically acceptable dosage form.

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