Soil sphingomonad c3 and application thereof

Abstract

This invention discloses a soil sphingopyxis terrae bacterium C3 and its applications. This soil sphingopyxis terrae bacterium C3 was deposited at the China Center for Type Culture Collection (CCTCC) on January 30, 2026, with accession number CCTCC NO: M 2026296. It can tolerate high concentrations (2000 mg / L) of meropenem and can efficiently degrade it as the sole carbon and energy source. Within 72 hours, it can degrade 50% of 2000 mg / L meropenem, and within 12 hours, the degradation rates for 4 mg / L and 50 mg / L meropenem can reach 100% and 70%, respectively. It has broad application prospects in the biological removal of meropenem from pharmaceutical wastewater, hospital wastewater, and urban sewage.

Description

Technical Field

[0001] This invention relates to a soil sphingopyxis terrae C3 strain and its application in degrading the antibiotic meropenem. Background Technology

[0002] Meropenem is a carbapenem antibiotic widely used to treat serious infections due to its broad-spectrum antibacterial activity and high resistance to β-lactamases. However, the increasing clinical use of meropenem has led to a growing risk of its residue remaining in the environment after being discharged into pharmaceutical wastewater, medical wastewater, and municipal sewage. Recent studies have shown that traditional wastewater treatment systems have limited removal efficiency for meropenem, allowing it to enter the environment and exert strong selective pressure on bacterial communities, promoting the emergence and spread of drug-resistant bacteria.

[0003] While chemical and physical methods can efficiently remove meropenem, these techniques are typically costly and can cause secondary pollution. In contrast, biodegradation using microorganisms is a more economical, environmentally friendly, and effective method. Studies have reported that some microorganisms, such as *Pseudomonas* and *Bacillus*, can degrade meropenem, but with low efficiency. Current research on the biodegradation of meropenem in natural environments is still limited. To date, there are no reports on the use of *Sphingopyxis terrae* in the removal of the antibiotic meropenem. Summary of the Invention

[0004] Objective of the Invention: In view of the problems existing in the prior art, one objective of the present invention is to provide a soil sphingopyxis terrae C3 bacterium, and another objective of the present invention is to provide the application of the soil sphingopyxis terrae C3 bacterium in the degradation of meropenem.

[0005] Technical Solution: This invention relates to a soil sphingopyxis terrae C3 bacterium, which was deposited at the China Center for Type Culture Collection (CCTCC) on January 30, 2026, with accession number CCTCC NO: M 2026296. The 16S rDNA sequence of soil sphingopyxis terrae C3 is shown in SEQ ID NO.3.

[0006] The present invention also includes a strain of fermentation broth comprising the soil sphingopyxis terrae C3 described in this invention.

[0007] The present invention also includes a microbial preparation comprising the soil sphingopyxis terrae C3 strain or the fermentation broth of the strain described in the present invention.

[0008] The present invention also includes the application of the soil sphingopyxis terrae C3, the fermentation broth of the strain described in the present invention, or the microbial preparation described in the present invention in the degradation of meropenem.

[0009] In this application, *Sphingopyxis terrae* C3 or its strain fermentation broth or microbial preparation is added to a solution containing meropenem. The meropenem-containing solution is either meropenem-containing wastewater or an aqueous solution containing meropenem and inorganic salts. The meropenem-containing wastewater type includes pharmaceutical wastewater, hospital wastewater, municipal sewage, or polluted surface water. The inorganic salts in the meropenem and inorganic salt aqueous solution are one or more of the following: K₂HPO₄, NaCl, NH₄Cl, KH₂PO₄, MgSO₄·7H₂O, CaCl₂·2H₂O, FeSO₄·7H₂O, MnSO₄·H₂O, CuCl₂H₂O, or NaMoO₄·2H₂O. Specifically, the concentrations include: K₂HPO₄ 1.5 g / L, NaCl 1.0 g / L, NH₄Cl 0.5 g / L, KH₂PO₄ 0.5 g / L, MgSO₄·7H₂O 0.2 g / L, CaCl₂·2H₂O 0.02 g / L, FeSO₄·7H₂O 1 mg / L, MnSO₄·H₂O 1 mg / L, CuCl₂H₂O 0.25 mg / L, and NaMoO₄·2H₂O 0.25 mg / L. The concentration of meropenem in the solution ranges from 0.001 to 2000 mg / L, and the amount of Sphingopyxis terrae C3 added is 1% to 10% based on the inoculum size at OD₆00=1.

[0010] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0011] (1) The soil sphingopyxis terrae C3 provided by the present invention has extremely strong meropenem tolerance and can grow normally and maintain degradation activity at a concentration of up to 2000 mg / L, which solves the pain point of difficult biological treatment of high-concentration pharmaceutical wastewater.

[0012] (2) The Sphingopyxis terrae C3 strain provided by this invention has excellent biodegradation performance for meropenem. It can achieve a degradation rate of 50% for 2000 mg / L meropenem within 72 hours, and a degradation rate of 100% and 70% for 4 mg / L and 50 mg / L meropenem within 12 hours, respectively. This can significantly improve the removal efficiency of meropenem in wastewater and reduce operating costs. Attached Figure Description

[0013] Figure 1 This is a colony diagram of Sphingopyxis terrae C3 bacteria in soil from Example 1;

[0014] Figure 2 This is a scanning electron microscope image of Sphingopyxis terrae C3 in Example 1.

[0015] Figure 3 This is a phylogenetic tree diagram of the soil sphingopyxis terrae C3 bacterium in Example 1;

[0016] Figure 4 The graph shows the degradation effect of soil sphingopyxis terrae C3 on different concentrations of meropenem in an inorganic salt culture medium matrix in Example 2.

[0017] Figure 5 This is a graph showing the changes in antibacterial activity of the products from the degradation of meropenem by Sphingopyxis terrae C3 in Example 2.

[0018] Figure 6 This is a diagram showing the degradation effect of meropenem by Sphingopyxis terrae C3 in wastewater matrix in Example 3. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0020] Example 1

[0021] 1. Isolation of soil sphingopyxis terrae C3

[0022] Sphingopyxis terrae C3 is a Gram-negative bacterium that was domesticated and isolated from aerobic activated sludge taken from a municipal wastewater treatment plant in Nanjing. The specific steps are as follows:

[0023] Add 100 mL of inorganic salt culture medium (composition: K₂HPO₄ 1.5 g / L, NaCl 1.0 g / L, NH₄Cl 0.5 g / L, KH₂PO₄ 0.5 g / L, MgSO₄·7H₂O 0.2 g / L, CaCl₂·2H₂O 0.02 g / L, FeSO₄·7H₂O 1 mg / L, MnSO₄·H₂O 1 mg / L, CuCl₂·H₂O 0.25 mg / L, NaMoO₄·2H₂O 0.25 mg / L) and 5 mL of activated sludge to a 250 mL Erlenmeyer flask, and add meropenem to bring the final concentration to 2 mg / L. Place the Erlenmeyer flask in a constant temperature shaker and incubate at 30°C. o C, rotation speed 180 rpm. After 7 days of culture, take 20 mL of mud-water mixture from the conical flask and add it to another conical flask containing 80 mL of fresh inorganic salt medium. Then add meropenem solution to a final concentration of 5 mg / L to complete one subculture enrichment. Repeat this subculture step 5 times, increasing the meropenem concentration in gradients of 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, and 100 mg / L to obtain enriched bacterial suspensions. After gradient dilution, inoculate the suspensions into 96-well plates for single-cell isolation. Select single-cell suspensions for streak plating. Figure 1 The selected candidate bacteria were added to an inorganic salt culture medium, and 20 mg / L (final concentration) of meropenem was added to conduct degradation performance tests. The strain with the highest degradation efficiency was selected, namely strain C3.

[0024] Strain C3 was observed using scanning electron microscopy. Figure 2 Its shape is rod-shaped, and its size ranges from approximately 0.5 µm × 1.5 µm to 0.46 µm × 2.0 µm.

[0025] 2. Molecular biological identification information and construction of phylogenetic tree

[0026] The strain C3 was identified using 16S rRNA gene sequencing. The 16S rRNA gene of strain C3 was amplified using bacterial culture PCR. The primer sequences used were: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 1); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2). The PCR reaction system consisted of: 15.0 μL 2×Taq® PCR MasterMix (TransGold, China), 11.0 μL sterile enzyme-free water, 1.0 μL (10 μM) forward primer, 1.0 μL (10 μM) reverse primer, and 2 μL bacterial culture. The PCR program was set as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 80 s, for a total of 32 cycles; 72℃ extension for 10 min.

[0027] The PCR products were purified using an agarose gel DNA purification kit (Takara, Japan), and the nucleotide sequence obtained by 16S rDNA sequencing is shown in SEQ ID NO. 3.

[0028] SEQ ID NO. 3:

[0029]

[0030] BLAST homology alignment of the above sequences in the NCBI-nr database showed that strain C3 had the highest similarity to *Sphingopyxis terrae* (see [link to BLAST analysis]). Figure 3 It was identified as *Sphingopyxis terrae* and named *Sphingopyxis terrae* C3. This strain was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China, on January 30, 2026, with accession number CCTCC NO: M 2026296.

[0031] Example 2

[0032] This embodiment provides the degradation efficiency of strain C3 obtained in Example 1 on different concentrations of meropenem, as well as the product bioactivity (antibacterial activity) test.

[0033] Preparation of strain C3 suspension: The strain C3 obtained in Example 1 was inoculated into LB liquid medium that had been sterilized by high temperature and high pressure, and cultured in a constant temperature shaker at 30°C and 160 rpm for 16 h. After the strain reached the logarithmic phase, the bacterial suspension was centrifuged at 8000×g for 5 min, the supernatant was discarded, and the precipitated bacterial cells were resuspended in inorganic salt medium (same as in Example 1). The above operation was repeated 3 times. Finally, the precipitated bacterial cells were resuspended in inorganic salt medium to obtain the C3 bacterial suspension for later use.

[0034] Meropenem Degradation Assay: To evaluate the tolerance threshold of strain C3 to meropenem and its biodegradation characteristics at high and low concentration gradients, degradation systems were constructed using nine meropenem concentration gradients: 4 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, and 2000 mg / L. Negative control groups without bacterial culture were also established at each concentration. In the experiment, 100 mL of autoclaved inorganic salt medium was added to a 250 mL Erlenmeyer flask, followed by a 10% inoculum of bacterial suspension with an OD600 of 1, ensuring consistent initial conditions for all groups. Meropenem was then added to the systems according to the experimental gradient, achieving final concentrations within the predetermined range of 4–2000 mg / L. All experimental systems were cultured in a constant-temperature shaker at 30℃ and 160 rpm. Samples were taken at 0 h, 12 h, 24 h, 36 h, 48 h, and 72 h. After filtration through a 0.22 μm aqueous filter membrane, the residual meropenem at each time point was quantitatively determined by high-performance liquid chromatography (HPLC, 1290 Infinity II, Agilent Technologies, USA). The HPLC column was a C18 column (4.6 × 100 mm, 5 μm, Agilent Technologies, USA). The detection conditions are shown in Table 1. The degradation effect is as follows: Figure 4 As shown.

[0035] Table 1 Detection conditions for high performance liquid chromatography

[0036]

[0037] Figure 4The results showed that strain C3 exhibited significant biodegradation ability at various concentration gradients of meropenem. In the low concentration range (4–25 mg / L), meropenem was almost completely removed within 12–24 h, indicating that this strain has extremely strong degradation ability under low load conditions, and the degradation process was not inhibited by drug concentration. For 4 mg / L meropenem, strain C3 could completely remove it within 12 hours. In contrast, the reported removal of only about 50% of 4 mg / L meropenem by *Pseudomonas putida* R51 within 72 hours (Chuanqing Z, Yingping Z, Jiafang F, et al. Cadmium stress efficiently enhanced meropenem degradation by the meropenem- and cadmium-resistant strain *Pseudomonas putida* R51[J]. *Journal of Hazardous Materials*, 2022, 429128354-128354. DOI:10.1016 / J.JHAZMAT.2022.128354.), indicating that strain C3 has a significant degradation advantage. When the concentration was increased to a moderate level (50 mg / L), the system still maintained a high degradation rate, with a significant decrease in concentration in the first 24 hours and a degradation rate of 100% after 48 hours. Under high concentration conditions (1000 mg / L and 2000 mg / L), although the overall degradation rate decreased, the degradation rate was still as high as 50-70% after 72 h, indicating that strain C3 has a high tolerance to meropenem.

[0038] The bioactivity (antibacterial activity) of the degradation products of the 50 mg / L concentration group within 48 h was tested using Escherichia coli DH5α (purchased from Sangon Biotech (Shanghai) Co., Ltd.), specifically determining the minimum inhibitory concentration (MIC, expressed here as the product dilution factor). The results are as follows: Figure 5 As shown. Figure 5 The results showed that in the degradation group without strain C3 (CK), the product consistently exhibited significant antibacterial activity, with a MIC of 1 / 4096. In contrast, the antibacterial activity of the product in the strain C3 degradation group decreased sharply with the degradation process, reaching only 1 / 16 by 24 h. These results indicate that strain C3 can not only efficiently degrade meropenem, but its degradation products also lose their viability and do not pose a risk of exerting selective pressure on microorganisms.

[0039] Example 3

[0040] This embodiment provides the degradation efficiency of meropenem by strain C3 in urban sewage substrate.

[0041] Since strain C3 can degrade meropenem using it as both a carbon source and an energy source, the degradation experiment was conducted in a municipal wastewater system to investigate whether the complex organic matrix would affect the degradation effect of strain C3.

[0042] Influent and effluent from the secondary sedimentation tank of a municipal wastewater treatment plant were collected and transported to the laboratory. After filtration through a 0.22 μm aqueous membrane to remove suspended particles and impurities, the filtrate was placed in 250 mL sterile Erlenmeyer flasks. Meropenem (final concentration) was added, followed by inoculation of a pre-prepared C3 bacterial suspension (OD600=1) at 10% (v / v) into the Erlenmeyer flasks (C3 bacterial suspension preparation was the same as in Example 2), establishing a degradation system with a final volume of 100 mL. The Erlenmeyer flasks were incubated in a constant temperature shaking incubator at 30 ℃ and 160 rpm. Samples were taken at 0 h, 12 h, 24 h, 36 h, 48 h, and 72 h of incubation. After filtration through a 0.22 μm aqueous membrane, the meropenem concentration in the samples was determined by HPLC. A blank (CK) without C3 bacterial suspension was used as a control. The HPLC detection method was the same as in Example 2, and the results are as follows. Figure 6 As shown.

[0043] Figure 6 The results showed that in actual wastewater systems, the addition of strain C3 completely removed 50 mg / L meropenem within 36 h. In contrast, the removal rate of the group without strain C3 was only 25% after 72 h. This indicates that strain C3 can still maintain high degradation activity for meropenem in complex organic matrix and has potential application value in treating antibiotic pollution in actual wastewater.

Claims

1. A soil sphingosine box bacterium ( Sphingopyxis terrae C3, characterized in that, The soil sphingosine box bacteria C3 was deposited at the China Center for Type Culture Collection on January 30, 2026, with accession number CCTCC NO: M 2026296.

2. The soil sphingosine box bacteria C3 according to claim 1, characterized in that, The 16S rDNA sequence of the soil sphingosine box bacteria C3 is shown in SEQ ID NO.

3.

3. A fermentation broth for a bacterial strain, characterized in that, Includes the soil sphingosine box bacteria C3 as described in claim 1 or 2.

4. A microbial preparation, characterized in that, The fermentation broth includes the soil sphingosine box bacteria C3 as described in claim 1 or 2, or the strain as described in claim 3.

5. The application of the soil sphingosine box bacteria C3 as described in claim 1 or 2, the fermentation broth of the strain as described in claim 3, or the microbial preparation as described in claim 4 in the degradation of meropenem.

6. The application according to claim 5, characterized in that, When applying, add the fermentation broth of *Sphingosine Box Bacterium* C3 or its strain, or its microbial preparation, to a solution containing meropenem.

7. The application according to claim 6, characterized in that, The concentration of meropenem in the solution is 0.001~2000 mg / L, and the amount of soil sphingosine box bacteria C3 added is 1%~10% based on the inoculum amount of bacteria with OD600=1.

8. The application according to claim 5, characterized in that, The meropenem-containing solution is either meropenem-containing wastewater or an aqueous solution containing meropenem and inorganic salts.

9. The application according to claim 8, characterized in that, Wastewater containing meropenem can be pharmaceutical wastewater, hospital wastewater, municipal sewage, contaminated surface water, or contaminated groundwater.

10. The application according to claim 8, characterized in that, The inorganic salt in the meropenem and inorganic salt aqueous solution is one or more of K2HPO4, NaCl, NH4Cl, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, FeSO4·7H2O, MnSO4·H2O, CuCl2·H2O, or NaMoO4·2H2O.

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