Bacteroides capillosus wj01 strain and application thereof

Abstract

The application discloses a Fibrobacter succinogenes WJ01 strain and application thereof, and belongs to the technical field of microorganisms. Cellulomonas taurus The strain is named WJ01, and is preserved in the China General Microbiological Culture Collection Center on June 15, 2026, with a preservation number of CGMCC NO.39234. The WJ01 strain of the application can absorb phosphorus elements in water bodies, and can significantly reduce the total phosphorus content in the water body environment, so that the purpose of treating total phosphorus pollution in the water body can be achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Cyclomonella tauri* WJ01 and its applications. Background Technology

[0002] Since the 1990s, with rapid economic and social development, multiple cascade hydropower stations have been developed and put into operation along the Guizhou section of the Wujiang River at various altitudes. This development has led to a series of environmental pollution problems. After the construction of reservoirs and dams, the slowing of water flow and the entry of various types of soil materials into the reservoirs have altered the pH and redox conditions of the water, changing the presence and migration capacity of certain heavy metals. This affects dissolved oxygen, oxygen consumption, pesticide degradation, and the activity of microorganisms in the water, causing variations in water quality. The Wujiangdu Reservoir suffers from severe total phosphorus contamination, exceeding the standard by 23.28-36.9 times, causing the water quality to deteriorate from Class II to Class V or worse. Furthermore, in the past 20 years, the rapid development of phosphate mining and phosphate chemical enterprises along the riverbanks of the Wujiang River has caused serious total phosphorus pollution. Therefore, how to control total phosphorus pollution in the Wujiang River basin has become an urgent problem to be solved. Summary of the Invention

[0003] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a strain of *Cyclomonella auricula-judae* WJ01 and its application, providing a microorganism that can absorb phosphorus from water, for the purpose of treating the ecological and environmental pollution problem of excessive total phosphorus in river water.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a strain of *Cytomyces cerevisiae* WJ01 is provided.

[0005] This invention has the following beneficial effects: This invention screened and obtained a strain capable of absorbing phosphorus from water. Through morphological and molecular biological identification, this strain was determined to be *Cyclomonella tamariscina*. Cellulomonas_taurus It was named WJ01 and deposited at the China General Microbiological Culture Collection Center on June 15, 2026, with accession number CGMCC NO.39234. Simulation results of actual water environment in the Wujiang River show that the WJ01 strain of this invention can absorb phosphorus from the water, significantly reducing the total phosphorus content in the aquatic environment, thereby achieving the goal of controlling total phosphorus pollution in the water. Detailed Implementation

[0006] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0007] Example 1: Isolation, screening and identification of phosphorus-absorbing microorganisms (1) Collection of phosphorus-contaminated soil samples: Based on the actual situation of water pollution in the cascade hydropower station area of ​​the Wujiang River Basin, soil samples were collected from the reservoir area of ​​the Silin Hydropower Station, which had the most severe total phosphorus pollution reported in previous studies, for microbial isolation and culture. During the sampling process, the covering materials such as branches and dead leaves on the surface of the selected tidal flat soil were removed first, and the soil drill was sterilized with 75% alcohol. Then, the top 0-15cm soil sample was drilled and sealed in a plastic bag for preservation. During transportation, the soil was preserved at low temperature using dry ice. After the samples were transported to the laboratory, they were briefly stored in a 4°C refrigerator, and subsequent sample dilution and microbial isolation and culture were carried out quickly.

[0008] (2) Soil sample dilution and microbial isolation and culture ① Weigh 10g of the soil sample collected in (1) using a balance, dissolve it in 90mL of sterile water, mix and shake well to obtain a 10⁻¹ dilution solution, then take 1mL of 10 -1 The diluted solution was mixed with 9 mL of sterile water to obtain a 10-fold dilution. -2 Prepare 10 dilution solutions sequentially, following the same principle. -3 10 -4 and 10 -5 Dilute the soil solution by a certain factor. Select 10. -2 10 -3 and 10 -4 Soil sample solutions of these three dilution gradients were used for fungal isolation and culture, with 10 being selected. -3 10 -4 and 10 -5 Soil sample solutions of these three dilution gradients were used for bacterial isolation and culture.

[0009] ② Bacterial isolation and culture were performed using NA medium and LB medium. 10 -3 10 -4 and 10 -5 Soil sample solutions of the three dilution gradients were evenly spread on NA and LB media, with three biological replicates for each dilution gradient. The samples were incubated at 28°C for 5 days in an inverted environment. Then, using an inoculation needle, single colonies of different morphologies and colors were picked and inoculated onto freshly prepared NA and LB media until single bacterial colonies were purified.

[0010] ③ Fungal isolation and culture were performed using PDA medium. 10 -2 10 -3 and 10 -4Soil sample solutions of the three dilution gradients were evenly spread on PDA medium, with three biological replicates for each dilution gradient. The samples were incubated at 28°C for 5 days in an inverted environment. Then, using an inoculation needle, single colonies of different colors and morphologies were picked and inoculated onto freshly prepared PDA medium until single fungal colonies were purified.

[0011] (3) Screening of phosphorus-absorbing microorganisms To further screen microorganisms that can absorb phosphorus from the solution, a basic phosphorus-free liquid culture medium was first prepared using the following culture medium formulation.

[0012] ① Basic phosphorus-free liquid culture medium for bacteria (taking 1000mL as an example, pH=6.5): MgSO4·7H2O 0.3g, KCl 0.2g, NH4NO3 1.0g, FeSO4·7H2O 0.01g, MnSO4·H2O 0.01g and sucrose 10g.

[0013] ② Basic phosphorus-free liquid culture medium for fungi (taking 1000mL as an example, pH=6.2): ​​MgSO4·7H2O 0.5g, KCl 0.5g, NH4NO3 3.0g, CaCl2·2H2O 0.1g, FeSO4·7H2O 0.01g, MgSO4·H2O 0.005g and sucrose 10g.

[0014] ③ According to the Environmental Quality Standard for Surface Water of the People's Republic of China (GB3838-2002), the threshold for total phosphorus content in Class V surface water (lake areas, reservoir areas) is greater than 0.2 mg / L. Taking into account the national standard threshold for total phosphorus content in surface water and the current status of total phosphorus pollution in the Siling section of the Wujiang cascade hydropower station, a liquid screening medium with approximately twice the national standard total phosphorus content (approximately 0.4 mg / L) was prepared using KH2PO4 on the basis of the above-mentioned phosphorus-free liquid culture medium. Then, the pH of the liquid culture medium was calibrated to the corresponding pH value (6.5 for bacteria and 6.2 for fungi) to obtain the screening medium. The prepared screening medium was then distributed and used for subsequent screening of phosphorus-absorbing microorganisms.

[0015] ④ Use an inoculation needle to pick up a single colony of the isolated and purified bacteria, mix it evenly with 30 mL of NB liquid medium, and then culture it at 28℃ and 180 rpm for constant temperature shaking to obtain the corresponding bacterial culture. Use PDB (potato dextrose broth) liquid medium for fungi, and perform the same operations as for bacteria to obtain the corresponding fungal culture.

[0016] ⑤ For bacteria, take 1 mL of the bacterial culture obtained from propagation and mix it evenly with 25 mL of bacterial selection medium; for fungi, take 1 mL of the fungal culture obtained from propagation and mix it evenly with 25 mL of fungal selection medium; set up 6 replicates for each bacterial culture; use 1 mL of NB liquid medium and 1 mL of PDB medium to replace the bacterial culture and fungal culture, respectively, and mix them with the corresponding bacterial and fungal selection medium as blank control groups, with 3 biological replicates for the control group.

[0017] ⑥ The well-mixed solution was incubated at 28℃ and 180 rpm for 4 days with constant temperature shaking. The cultured solution was then centrifuged at 5000 rpm for 10 min using a high-speed centrifuge to precipitate the strain and obtain the supernatant. The total phosphorus content of the supernatant was measured using the ammonium molybdate spectrophotometric method (GB11893-89). By comparing with the blank control, microorganisms that can significantly absorb phosphorus were screened. The screened microorganisms were then sent to Meiji Biotechnology Co., Ltd. (Shanghai) for strain identification.

[0018] 1. Morphological and molecular biological identification results The single colony morphology of strain WJ01 is round, 1-2 mm in diameter, with neat and regular edges; the center of the colony is raised, the colony color is pale yellow, the surface is smooth, moist, shiny, and translucent to slightly transparent, the colony is easy to pick up, the texture is soft, creamy, and without mucus.

[0019] The two ends of the Sanger sequencing sequence were subjected to quality control to remove low-quality bases, resulting in a clean sequence. This clean sequence was then assembled to obtain an assembled sequence (16S rRNA sequence, its nucleotide sequence is shown in SEQ ID NO.1). This assembled sequence was compared with the NT database, and the species with the highest similarity was selected as the bacterial identification result. Primer sequences are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3).

[0020] The strains screened in this invention were compared with the NT database. Cellulomonas_taurus The alignment rate was 99.639%. According to the sequencing results, its gene sequence is 1517bp (as shown in SEQ ID NO.1), and it is named WJ01.

[0021] 2. Phosphorus degradation capacity test results According to the requirements of "Water Quality - Determination of Total Phosphorus - Ammonium Molybdate Spectrophotometric Method" (GB11893-89), a standard working curve was plotted, and the test results are shown in Table 1.

[0022] Table 1 Determination of Total Phosphorus

[0023] A linear fit was performed on the absorbance of P and the concentration of P, and the fitting result is as follows: y = 2.3136x + 0.008; goodness of fit R0 2 =0.9998.

[0024] The total phosphorus determination results of the screening medium control group and strain WJ01 are shown in Table 2.

[0025] Table 2. Total phosphorus determination results for the control group and strain WJ01

[0026] The total phosphorus concentration measured after adding strain WJ01 and the control group were subjected to an independent samples t-test, and the statistical results are shown in Table 3.

[0027] Table 3. Results of Independent Samples t-test

[0028] The above results indicate that adding the WJ01 strain of this invention to a liquid solution with total phosphorus exceeding the national standard can significantly reduce the total phosphorus content in the liquid (P < 0.001), thereby achieving the goal of reducing total phosphorus pollution.

[0029] Example 2: Simulation Experiment of Actual Water Environment in the Wujiang River To further verify the phosphorus degradation ability of the strains screened in this invention in a real aquatic environment, 10 strains prepared during the microbial isolation and purification process were used. -1The soil solution was diluted and allowed to stand at room temperature for 24 hours. The supernatant was collected as a sample simulating the Wujiang River water environment. Single colonies of the isolated and purified bacteria were picked using an inoculation needle and thoroughly mixed with 30 mL of NB (Nutrient Broth) liquid medium. The mixture was then incubated at 28°C with constant temperature shaking at 180 rpm to amplify the bacterial culture, which served as the experimental group (6 biological replicates). 1 mL of the amplified bacterial culture was mixed thoroughly with 25 mL of the prepared simulated water sample. 1 mL of NB liquid medium was used instead of the bacterial culture and mixed with 25 mL of the prepared simulated water sample. The control group had 3 biological replicates. Then, the mixture was cultured at 28℃ and 180rpm for 4 days with constant temperature shaking. The cultured mixture was centrifuged at 5000rpm for 10min using a high-speed centrifuge to precipitate the solid and obtain the supernatant. The total phosphorus content of the supernatant was measured using the ammonium molybdate spectrophotometric method (GB11893-89). The ability to absorb phosphorus in a simulated water environment was determined by comparing it with the blank control.

[0030] The results of the actual water body simulation are shown in Table 4.

[0031] Table 4. Simulation results of actual water bodies

[0032] The t-test results for the experimental and control groups are shown in Table 5.

[0033] Table 5. t-test results for the experimental and control groups.

[0034] The above results indicate that, in a simulated real water environment, the WJ01 strain of this invention significantly reduces total phosphorus compared to the control group. However, due to limitations such as carbon source constraints, its effect on reducing total phosphorus in a simulated real water environment is slightly lower than that in a laboratory environment.

[0035] The 16S rRNA sequence of strain WJ01 in this invention is as follows:

[0036] 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 species of *Cyclomys* ( Cellulomonas_taurus strain WJ01, characterized in that, The WJ01 strain was deposited on June 15, 2026, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.39234.

2. The application of the WJ01 strain according to claim 1 in the preparation of a formulation for treating total phosphorus pollution in water bodies.

3. A preparation for treating total phosphorus pollution in water bodies, characterized in that, It includes at least one of the WJ01 strain as described in claim 1, its fermentation broth, and its fermentation supernatant.

4. The application of the WJ01 strain according to claim 1 in the treatment of total phosphorus pollution in water bodies.

5. The application of the formulation according to claim 3 in the treatment of total phosphorus pollution in water bodies.

6. A method for treating total phosphorus pollution in water bodies, characterized in that, Treatment is carried out by spraying the WJ01 strain of claim 1 or the preparation of claim 3 into the polluted water body.

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