Pseudomonas degrading chloramphenicol and application thereof

Abstract

The application discloses a pseudomonas for degrading chloramphenicol and application thereof. Pseudomonas The strain is named as Pseudomonas sp. MH-W1, the preservation number is GDMCC No: 66907, and the strain is preserved in the Guangdong Microbial Culture Collection Center on September 1, 2025. The strain can tolerate 200 mg / L chloramphenicol; and can degrade 100 mg / L chloramphenicol in 8 hours, and the degradation rate is about 80%. The method provided by the application can significantly shorten the degradation time and improve the degradation efficiency, the degradation condition is mild, the operation is simple, large-scale application is easy to realize, and the method has a wide industrialization popularization prospect. The strain is an aerobic bacterium, has fast growth and reproduction, is simple to prepare, low in cost, convenient to use and high in efficiency, and can be used for repairing chloramphenicol contaminated environment.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of environmental pollution treatment of wastewater, sewage, sludge, etc., and particularly relates to a pseudomonas for degrading chloramphenicol and application thereof. BACKGROUND

[0002] Chloramphenicol is a chlorinated nitro aromatic broad-spectrum antibiotic, which has been widely used in human medical treatment, veterinary medicine, animal husbandry and aquaculture fields. The antibiotic has good antibacterial activity and clinical efficacy, but due to its stable chemical structure, strong environmental persistence and high potential toxicity, it has been proved to have serious impact on the ecological system and human health. Chloramphenicol in the environment mainly comes from medical wastewater, livestock and aquaculture discharge and food residues, and its detection concentration ranges from ng·L -1 to mg·L -1 . The concentration of chloramphenicol in some sewage (wastewater) even exceeds the tolerance threshold of conventional microorganisms, significantly inhibiting their metabolic and degradation activity. Therefore, screening a tolerant strain that can still maintain high efficiency and rapid degradation capacity in a high-concentration chloramphenicol environment has become a key technical problem that needs to be solved in the field of antibiotic pollution bioremediation.

[0003] At present, the main methods for removing chloramphenicol residues include photocatalytic method, Fenton method, electrochemical degradation method and microbial degradation method, etc. Among them, microbial remediation is concerned due to its fast degradation rate, strong environmental compatibility and low cost. More importantly, microorganisms can completely mineralize or transform chloramphenicol into low-toxicity intermediates, thereby effectively reducing its environmental risk. Studies have shown that both microbial communities and pure culture strains have certain chloramphenicol degradation capacity. For example, the enriched bacterial community CL and CH can completely degrade 120 mg·L -1 of CAP in 5 days; the activated sludge bacterial community can remove 50 mg·L -1 of chloramphenicol in 24 h (Ma X, Qi M, Li Z, et al. Characterization of an efficient chloramphenicol-mineralizing bacterial consortium[J]. Chemosphere, 2019, 222: 149-155). Some single strains also show strong degradation capacity, such as strain CAP-1, which can degrade 50 mg·L -1 of CAP in 30 h (Ma X, Liang B, Qi M, et al. Novel Pathway for Chloramphenicol Catabolism in the Activated Sludge Bacterial IsolateSphingobium sp. CAP-1[J]. Environmental Science & Technology: ES&T, 2020(12):54, 7591-7600.); Sphingosomalidone CL5.1 can completely remove 120 mg·L within 48 h. -1 The CAP mineralization rate reached 50.4% (Zhang J, Gan W, Zhao R, et al. Chloramphenicol biodegradation by enriched bacterial consortia and isolated strain). Sphingomonas sp. CL5.1: Thereconstruction of a novel biodegradation pathway[J]. Water Research, 2020,187:116397.); Sphingomyelin Bacillus WTD-1 can degrade 100 mg·L within 60 h. -1 of chloramphenicol (Gao Y, Chen Y, Zhu F, et al. Revealing the biological significance of multiple metabolic pathways of chloramphenicol by Sphingobium sp. WTD-1[J]. Journal of Hazardous Materials, 2024, 469, 134069.). In addition, strains such as Citrobacter SZW2, Klebsiella YB1, and Bordetella C3 showed similar results at lower concentrations (10 mg·L⁻¹). -1 Under these conditions, after 2–10 days of treatment, its degradation efficiency ranged from 22.4% to 85.7% (Luo Y, Tan Z. The metabolic fate and detoxifying biotransformation of chloramphenicol by Citrobacter sp. SZW2[J]. Journal of Water Process Engineering, 2024, 105802.; Tan ZW, Yang XY, Liu YL, et al. Thecapability of chloramphenicol biotransformation of KlebsiellaWang Y, Luo Y, Tan Z, et al. Biotransformation of chloramphenicol by enriched bacterial consortia and the newly isolated bacterial strain Pseudomonas sp. YB1 under cadmium stress and its genome analysis. Chemosphere, 2023, 313, 137375.;Wang Y, Luo Y, Tan Z, et al. Biotransformation of chloramphenicol by enriched bacterial consortia and the newly isolated bacterial strain Bordetella sp.C3: Detoxifying biotransformation pathway and its potential application in agriculture[J]. Bioresource Technology, 2025, 415, 131713. However, the existing degrading strains generally have problems such as slow degradation rate, incomplete mineralization and insufficient tolerance to high-concentration pollutants, which limit their popularization and application in practical engineering. Therefore, there is still a key demand for developing new tolerant strains that can maintain high-efficiency degradation performance in high-concentration chloramphenicol contaminated environment. SUMMARY

[0004] The primary purpose of the present application is to provide a Pseudomonas that can efficiently and quickly degrade chloramphenicol in view of the low degradation rate and insufficient tolerance in the prior art.

[0005] Another purpose of the present application is to provide applications of the Pseudomonas that can efficiently and quickly degrade chloramphenicol.

[0006] To achieve the above purposes, the present application provides the following technical solutions:

[0007] A Pseudomonas that can efficiently and quickly degrade chloramphenicol, named Pseudomonas sp. Pseudomonas (Pseudomonas sp.) MH-W1, with the accession number GDMCC No: 66907, was deposited at the Guangdong Microbial Culture Collection Center of the Guangdong Institute of Microbiology, located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, on September 1, 2025.

[0008] The colony of the Pseudomonas is round, white and dry in texture, and the cells are oval and gram-negative.

[0009] A microbial inoculant for degrading chloramphenicol, comprising the Pseudomonas that can efficiently and quickly degrade chloramphenicol.

[0010] The microbial agent used to degrade chloramphenicol is the fermentation broth, spray-dried powder, or lyophilized powder of the above-mentioned highly efficient and rapid chloramphenicol-degrading Pseudomonas.

[0011] The microbial agent for degrading chloramphenicol is preferably prepared by the following steps:

[0012] (1) The above-mentioned efficient and rapid chloramphenicol-degrading Pseudomonas aeruginosa was cultured in a bacterial culture medium at a temperature of 27-32°C and in a shake flask culture or fermenter fermentation to obtain the fermentation broth of Pseudomonas aeruginosa.

[0013] (2) Separate the solid and liquid of the fermentation broth, and spray dry or freeze dry the obtained solid to obtain spray-dried powder or freeze-dried powder.

[0014] The bacterial culture medium preferably has the following composition: 10-15 g / L tryptone, 5-10 g / L yeast extract, 5-10 g / L sodium chloride, pH=6.8-7.4; more preferably: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH=7.0.

[0015] The preferred rotation speed for the shake flask culture is 100–250 rpm; more preferably 150–200 rpm.

[0016] The fermentation process in the fermenter is a conventional technique in the field, including the following steps: strain activation, stepwise scaling-up culture, and fermenter aeration culture.

[0017] The extent of the progressive scaling-up cultivation is determined based on the amount of seed required for the fermenter.

[0018] The above-mentioned efficient and rapid chloramphenicol-degrading Pseudomonas bacteria or microbial agents for chloramphenicol degradation are used in the remediation of chloramphenicol-containing environments.

[0019] The environment is at least one of soil and water.

[0020] The present invention has the following advantages and effects compared with the prior art:

[0021] The Pseudomonas aeruginosa provided by this invention ( Pseudomonas The MH-W1 strain (sp.) can tolerate 200 mg / L chloramphenicol and degrade 100 mg / L chloramphenicol within 8 hours, achieving a degradation rate of approximately 80%. This aerobic bacterium exhibits rapid growth and reproduction, is simple to prepare, low in cost, convenient to use, and highly efficient, making it suitable for the efficient biological treatment of chloramphenicol-containing waste and wastewater, and possessing good industrialization potential. Compared with previously reported chloramphenicol-degrading strains, Pseudomonas sp .MH-W1 achieved higher degradation rate in a shorter time, and had good high-concentration tolerance, showing significant technical progress. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1 is a scanning electron microscope (SEM) photo of Pseudomonas MH-W1.

[0023] Figure 2 Figure 2 is a phylogenetic tree of Pseudomonas MH-W1 constructed based on 16S rRNA gene sequence.

[0024] Figure 3 Figure 3 is a graph of the effect of different chloramphenicol concentrations on the degradation of Pseudomonas MH-W1.

[0025] Figure 4 Figure 4 is a schematic diagram of the effect of different chloramphenicol concentrations on the degradation rate and degradation rate of Pseudomonas MH-W1.

[0026] Figure 5 Figure 5 is a comparison graph of the degradation effect of different Pseudomonas MH-W1 and DSM 288 on chloramphenicol.

[0027] Figure 6 Figure 6 is a schematic diagram of the degradation rate and degradation rate of different Pseudomonas MH-W1 and DSM 288. DETAILED DESCRIPTION

[0028] The present application will be further described in detail below in conjunction with the examples and drawings, but the embodiments of the present application are not limited thereto.

[0029] Composition of LB liquid medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, pH = 7.0, solvent is deionized water.

[0030] The LB solid medium is 20 g / L agar added to the LB liquid medium.

[0031] Example 1: Collection, isolation and purification process of Pseudomonas MH-W1

[0032] Strain source: The sample was collected from the Pearl River natural water body (113°31'9"E, 22°59'2"N). When sampling, 50 mL polypropylene centrifuge tubes were used to collect water samples, and the samples were temporarily stored in sterilized vials.

[0033] Take 5.00 mL of the above fresh water sample into 50 mL of sterilized LB liquid medium containing 100 mg / L chloramphenicol (initial pH 7.0), and continuously cultivate at 30 ℃ for 3 d; then take 5% (v / v) supernatant to transfer to fresh LB medium containing 100 mg / L chloramphenicol, and cultivate at the same conditions for 2 d. Repeat the transfer for 2 times to enrich the bacterial population.

[0034] Use the dilution coating method to separate the strains, and use sterile distilled water to dilute the enrichment liquid to a concentration of 10 -3 ~10 -6 times in stages, take 0.1 mL of the diluted liquid to coat on the LB solid plate containing 100 mg / L chloramphenicol, use a sterile coating rod to evenly coat, and then cultivate in a 30 ℃ incubator for 2 d, pick single colonies to streak and purify, cultivate in the LB solid plate containing 100 mg / L chloramphenicol, and repeat the streaking and separation until a pure culture of single colonies is obtained.

[0035] Example 2: Identification of Pseudomonas MH-W1

[0036] The fastest growing purified strain colony obtained in Example 1 is round, white, and dry in texture, which is inoculated into sterile LB liquid medium containing 100 mg / L chloramphenicol, and cultivated at 30 ℃, 150 rpm, until the bacterial liquid grows to the logarithmic growth phase (OD 600 ≈3.0).

[0037] Take 5 mL of the logarithmic growth phase bacterial liquid into a 15 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Add about 3 mL of phosphate buffer containing 2.5% v / v glutaraldehyde (0.1 M, pH=7.4) to about 0.1 mL of bacterial cells, resuspend thoroughly, and then transfer into a new 15 mL centrifuge tube, and fix at 4 ℃ for 4 h. Then centrifuge at 8000 rpm for 5 min, discard the supernatant, and wash with 0.1 M phosphate buffer for 3 times. Use 30%, 50%, 70%, 85%, 90% and 100% (twice) ethanol solutions in volume percentage for gradient dehydration, each time for 15 min, all centrifuged at 8000 rpm for 5 min and then the supernatant was discarded. Then replace the ethanol with tert-butyl alcohol, stand for 20 min, and centrifuge again to discard the liquid. The obtained bacterial cell samples were frozen at -20 ℃ and -80 ℃ for 12 h respectively, and then freeze-dried for 12 h by a freeze dryer, and the prepared samples were observed under a scanning electron microscope (SEM5000X, Guoyili Quantum Technology Co., Ltd., China), and the results are shown in Figure 1 The bacterial cells obtained by screening are oval in shape, with a diameter of about 2-3 μm. Gram staining of the bacteria shows that the bacteria are gram-negative bacteria.

[0038] Take 1 mL of bacterial culture in the logarithmic growth phase into a centrifuge tube, centrifuge at 8000 r / min for 5 min, discard the supernatant, and wash once with 1 mL of sterile water. Perform PCR amplification using 16S rDNA gene primers 27F and 1492R (purchased from Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.). The PCR amplification system included: 12.5 μL Tap DNA mix, 1 μL resuspended bacterial culture, 0.5 μL of 10 μM primer 27F, 0.5 μL of 10 μM primer 1492R, and 10.5 μL ddH2O. The PCR reaction program was: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15 s, annealing at 55 ℃ for 15 s, extension at 72 ℃ for 60 s, 30 cycles; final extension at 72 ℃ for 5 min. PCR products were detected by agarose gel electrophoresis to determine whether the target band was obtained. The qualified products were sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for sequencing. The sequencing results were then subjected to BLAST alignment and phylogenetic tree analysis. The results are as follows: Figure 2 As shown, the screened strain belongs to the genus *Pseudomonas* and is named... Pseudomonas sp. MH-W1. This strain was deposited on September 1, 2025, at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66907.

[0039] Primer 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′;

[0040] Primer 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′.

[0041] 16S rDNA sequence:

[0042]

[0043] Example 3: Test of the ability of Pseudomonas MH-W1 to degrade chloramphenicol

[0044] First, *Pseudomonas MH-W1* seed culture was cultured in LB liquid medium under the same conditions as in Example 2. The culture was then cultured until the optical density (OD) of the culture medium was reached. 600 When the pH reaches around 3.0, the seed culture of strain MH-W1 is obtained.

[0045] MH-W1 seed culture was inoculated into LB medium at pH 7 at an inoculation rate of 1% (v / v). The initial concentrations of chloramphenicol in the medium were set at 10 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L, respectively. The culture was carried out at 30℃ and 150 r / min under shaking conditions. Samples were taken at 0 h, 2 h, 4 h, and 8 h, and after centrifugation and filtration, the residual concentration of chloramphenicol in the supernatant was determined using a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II system, USA).

[0046] The results are as follows Figure 3 As shown, strain MH-W1 exhibited significant degradation ability at different initial chloramphenicol concentrations, with the chloramphenicol concentration continuously decreasing with prolonged culture time. Specifically, at an initial concentration of 150 mg / L, the degradation rate reached 75.35% after 8 h. Figure 4 The corresponding degradation rate was 14.82 mg / L·h. -1 Furthermore, although the overall degradation rate decreased slightly with increasing initial concentration, the degradation rate showed an increasing trend, indicating that this strain has strong tolerance and degradation ability to high concentrations of chloramphenicol. In summary, these results demonstrate that *Pseudomonas MH-W1* possesses excellent chloramphenicol tolerance and rapid degradation performance, and can efficiently remove high concentrations of chloramphenicol in a short time, showing high application potential.

[0047] Example 4: Comparison of the degradation abilities of two Pseudomonas strains, MH-W1 and DSM 288, on chloramphenicol.

[0048] Based on the 16S rDNA phylogenetic tree results, *Pseudomonas aeruginosa* MH-W1 is most closely related to *Pseudomonas aeruginosa* in evolutionary terms. *Pseudomonas aeruginosa* was selected as the primary strain. Pseudomonas aeruginosaDSM 288 (purchased from the German Culture Collection of Microorganisms, DSMZ) was used as a control strain to assess the species-wide applicability of chloramphenicol degradation ability. DSM288 was inoculated into LB broth at a seed culture inoculation rate of 5% (v / v) and cultured aerobically in 150 mL Erlenmeyer flasks at 30°C and 150 rpm for 16 h with shaking. The culture was analyzed when the optical density (OD) of the culture medium was reached. 600 When the pH reached approximately 3.0, seed culture of strain DSM 288 was obtained. The obtained DSM288 seed culture was inoculated into LB liquid medium at pH 7.0 at an inoculum rate of 1% (v / v), with the initial chloramphenicol concentration set at 100 mg / L. The culture was carried out at 30 ℃ and 150 rpm, and samples were taken at 0 h, 2 h, 4 h, and 8 h. After centrifugation and filtration, the residual chloramphenicol concentration in the supernatant was determined using high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity II system, USA).

[0049] The seed culture preparation conditions for strain MH-W1 were the same as in Example 3. The obtained MH-W1 seed culture was inoculated into LB medium at pH 7.0 at an inoculum rate of 1% (v / v), with an initial chloramphenicol concentration of 100 mg / L. The culture was carried out at 30 °C and 150 r / min, and samples were taken at 0 h, 2 h, 4 h, and 8 h. After centrifugation and filtration, the residual concentration of chloramphenicol in the supernatant was determined using high-performance liquid chromatography (Agilent 1260 Infinity II system, USA).

[0050] The results showed that strain DSM 288 did not exhibit significant degradation ability at an initial chloramphenicol concentration of 100 mg / L, and the chloramphenicol concentration remained essentially unchanged with prolonged culture time; while strain MH-W1 showed higher degradation activity under the same conditions. Figure 5 Within 8 hours, the degradation rates of chloramphenicol by strains DSM288 and MH-W1 were 21.34% and 80.03%, respectively. Figure 6 The corresponding degradation rates were 2.70 mg / L·h. -1 and 10.03 mg / L·h -1 In summary, the chloramphenicol degradation rate of Pseudomonas MH-W1 is approximately 3.7 times that of DSM 288, indicating that the highly efficient chloramphenicol degradation ability of Pseudomonas MH-W1 is unique.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Pseudomonas aeruginosa that degrades chloramphenicol, characterized in that: The name of the Pseudomonas species that degrades chloramphenicol is Pseudomonas (…). Pseudomonas sp.) MH-W1, with accession number GDMCC No: 66907, was deposited on September 1, 2025 at the Guangdong Provincial Microbial Culture Collection Center, Guangdong Academy of Sciences Institute of Microbiology, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A microbial agent for degrading chloramphenicol, characterized in that: The Pseudomonas aeruginosa comprising the chloramphenicol-degrading strain of claim 1.

3. The microbial agent for degrading chloramphenicol according to claim 2, characterized in that: The microbial agent for degrading chloramphenicol is the fermentation broth, spray-dried powder, or freeze-dried powder of the Pseudomonas aeruginosa that degrades chloramphenicol as described in claim 1.

4. The microbial agent for degrading chloramphenicol according to claim 3, characterized in that: The microbial agent for degrading chloramphenicol is prepared through the following steps: (1) The Pseudomonas aeruginosa of claim 1, which degrades chloramphenicol, is cultured in a bacterial culture medium at a temperature of 27-32°C and in a shake flask culture or a fermenter fermentation to obtain the fermentation broth of Pseudomonas aeruginosa. (2) Separate the solid and liquid of the fermentation broth, and spray dry or freeze dry the obtained solid to obtain spray-dried powder or freeze-dried powder.

5. The microbial agent for degrading chloramphenicol according to claim 4, characterized in that: The bacterial culture medium is composed of the following: 10-15 g / L tryptone, 5-10 g / L yeast extract, 5-10 g / L sodium chloride, pH=6.8-7.

4.

6. The application of the Pseudomonas aeruginosa of claim 1 that degrades chloramphenicol or the microbial agent for degrading chloramphenicol of any one of claims 2 to 5 in the remediation of chloramphenicol-containing environments.

7. The application according to claim 6, characterized in that: The environment is at least one of soil and water.

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