Chryseobacterium sp. For degrading chloramphenicol and application thereof

Abstract

The invention discloses a strain of Chryseobacterium sp. For degrading chloramphenicol and application thereof. Belongs to the technical field of biochemical environment treatment. The chloramphenicol degrading bacterium provided by the invention is identified as Chryseobacterium sp. Through 16S rDNA (ribosomal deoxyribonucleic acid), the strain is preserved in the China Center for Type Culture Collection on November 26, 2024, and the preservation number is CCTCC NO: M 20242642. According to the efficient chloramphenicol degrading bacterium, the degradation rate of a degrading bacterium agent prepared from the efficient chloramphenicol degrading bacterium on chloramphenicol with the initial concentration of 100 mg / L within 48 h is as high as 96%. The chloramphenicol degrading bacterial agent is suitable for treating industrial wastewater, pharmaceutical wastewater, fishpond culture wastewater and other wastewater containing chloramphenicol pollution, the degrading effect is remarkable, the speed is high, secondary pollution can be avoided, and an efficient, economical and environment-friendly technical means is provided for solving the problem of antibiotic pollutant treatment.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical environment treatment, and more specifically to a Chryseobacterium chlororaphene-degrading strain and application thereof. Background Art

[0002] Chloramphenicol is a broad-spectrum antibiotic that is widely used in the treatment of infections in humans and animals because of its strong antibacterial and bactericidal effects. However, due to its stable structure and difficulty in degradation, it is easy to enter the environment through excretion, especially forming long-term residues in water bodies. At present, the environmental pollution problem of chloramphenicol has received widespread attention, especially in aquaculture wastewater, pharmaceutical industrial wastewater and hospital discharge, where its residual concentration often reaches above the ecological safety threshold. These residual chloramphenicol will not only directly damage the health of animals and humans, such as causing hematopoietic dysfunction, immunosuppression and other toxic reactions, but may also induce pathogens to develop drug resistance through long-term low-concentration exposure, thereby posing a major threat to public health and the medical system. In addition, chloramphenicol contains amide groups and dichloroethanol groups, which make it have high chemical stability and biological persistence in the environment, making it difficult to effectively remove it by conventional physical and chemical treatment methods, further exacerbating its environmental accumulation and ecological risks. Therefore, the development of efficient and green chloramphenicol degradation technology to reduce its harm to the ecological environment and human health has become an important scientific issue that needs to be solved urgently.

[0003] At present, the treatment of chloramphenicol wastewater mainly utilizes bio-electrochemical system reduction degradation, radiation degradation and ozone oxidation degradation. Compared with the physicochemical method, the biological method degrades antibiotics through the metabolic activity of microorganisms or the catalytic action of enzymes secreted by microorganisms. Not only does it require milder reaction conditions, it can effectively remove antibiotics from the environment and will not cause secondary pollution to the environment. In biological treatment, the screening and application of chloramphenicol degradation strains is one of the key links. Studies have shown that the degradation effect of chloramphenicol degradation mixed bacteria screened by sludge as the bacterial source is good. When the chloramphenicol concentration is 50 mg / L, the chloramphenicol mineralization rate is 71.50% in 5 days; the chloramphenicol degradation bacteria screened from feces have a degradation rate of 84.00% for 55 mg / L chloramphenicol at 12 days; the chloramphenicol degradation bacteria screened from river water have a removal rate of 89.54% for chloramphenicol at a chloramphenicol concentration of 50 μg / L by Pseudomonas putida, while the removal rate of chloramphenicol by Aeromonas hydrophila is only 13.79%. These studies show that the ability of specific strains to degrade chloramphenicol varies greatly, which is affected by factors such as strain characteristics, environmental conditions and chloramphenicol concentration. It can be seen that although there are chloramphenicol degrading bacteria with certain effects in the prior art, their degradation conditions are relatively high, which limits their wide application in wastewater treatment.

[0004] In summary, how to provide a chloramphenicol-degrading bacterial agent that is highly efficient, adaptable and easy to use is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a Chryseobacterium tumefaciens strain capable of degrading chloramphenicol and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strain of Chryseobacterium that degrades chloramphenicol, the Chryseobacterium being named 41012, and being classified as Chryseobacterium sp., was deposited in the China Center for Type Culture Collection on November 26, 2024, with the deposit number CCTCC NO:M 20242642, and the deposit address is Wuhan University, Wuhan, China.

[0008] The above-mentioned application of Chryseobacterium tumefaciens in degrading chloramphenicol.

[0009] The application of the above-mentioned Chryseobacterium spp. in the preparation of a bacterial agent for degrading chloramphenicol.

[0010] The above-mentioned application of Chryseobacterium tumefaciens in treating chloramphenicol-contaminated wastewater.

[0011] Furthermore, the wastewater is industrial wastewater, aquaculture wastewater, or pharmaceutical wastewater.

[0012] A chloramphenicol-degrading bacterial agent comprises the above-mentioned Chryseobacterium.

[0013] Further, the preparation method is as follows:

[0014] The Chryseobacterium spp. is cultured in a beef extract peptone liquid culture medium.

[0015] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0016] (1) The present invention provides a chloramphenicol efficient degradation bacterium Chryseobacterium sp.41012. The degradation rate of the microbial agent prepared by using the microbial agent for 100 mg / L chloramphenicol is 96%, which provides rich microbial resources for the treatment of chloramphenicol wastewater and is of great significance to environmental protection and water resource utilization.

[0017] (2) Chryseobacterium sp. 41012 in the present invention is a single degrading bacterium for chloramphenicol degradation, which is simple to prepare, convenient to use and highly effective, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 This is a colony morphology diagram of the strain Chryseobacterium sp.41012 in Example 1 of the present invention;

[0020] Figure 2 This is a transmission electron micrograph of the bacterial cell of the strain Chryseobacterium sp.41012 in Example 1 of the present invention;

[0021] Figure 3 is the phylogenetic tree of strain Chryseobacterium sp.41012 in Example 1 of the present invention;

[0022] Figure 4 This is a schematic diagram showing the effect of different carbon sources on the degradation of chloramphenicol by strain Chryseobacterium sp.41012 in Example 4 of the present invention;

[0023] Figure 5 This is a schematic diagram showing the effect of different glucose addition amounts on the degradation of chloramphenicol by strain Chryseobacterium sp.41012 in Example 4 of the present invention;

[0024] Figure 6 This is a schematic diagram showing the effect of different chloramphenicol concentrations on the degradation of chloramphenicol by strain Chryseobacterium sp.41012 in Example 4 of the present invention;

[0025] Figure 7 This is a schematic diagram showing the effect of different pH values ​​on the degradation of chloramphenicol by strain Chryseobacterium sp.41012 in Example 4 of the present invention;

[0026] Figure 8 Schematic diagram of the effect of different temperatures on the degradation of chloramphenicol by strain Chryseobacterium sp.41012 in Example 4 of the present invention;

[0027] Fig. 9 These are the actual application results of the bacterial agent of the highly efficient chloramphenicol-degrading bacteria Chryseobacterium sp. 41012 in Examples 5 to 7 of the present invention in the treatment of chloramphenicol-containing wastewater. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0030] Example 1

[0031] Isolation and identification of highly efficient chloramphenicol-degrading bacterium Chryseobacterium sp.41012

[0032] Source of strain: activated sludge from treating chloramphenicol wastewater.

[0033] Separation process:

[0034] 1) Initial screening

[0035] Weigh 10g of the above activated sludge into 90mL of screening medium, culture at 28℃ and 120r / min for 2 days, and let it stand for 1h. -1 ~10 -8 0.1 mL of bacterial suspension was applied to beef extract peptone solid medium (containing 100 mg / L chloramphenicol) and cultured at 28°C for 2-3 days. The beef extract peptone solid medium without bacteria was used as a blank control, and 10 strains of bacteria were initially screened.

[0036] The screening medium composition was: glucose 0.281 g / L, NH4Cl 0.229 g / L, K2HPO4 0.067 g / L, MgSO4·7H2O 0.049 g / L, CaCl2 0.023 g / L, trace elements 1 mL / L (H3BO3 0.11 g / L, CuSO4·5H2O 0.22 g / L, FeCl3·6H2O 0.24 g / L, MnCl4·H2O 0.20 g / L, ZnSO4·7H2O 0.10 g / L, NaMoO4·2H2O 0.11 g / L, CoCl2·6H2O 0.20 g / L), pH 7.5, and sterilized at 115°C for 30 min.

[0037] The beef extract peptone solid culture medium is composed of: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, distilled water 1000mL, agar powder 13.5g / L, pH 7.0-7.2, and high pressure sterilization at 121°C for 20min.

[0038] 2) Rescreening

[0039] Pick appropriate amount of colonies of the 10 strains of bacteria obtained in the initial screening, inoculate them into beef extract peptone liquid culture medium, culture them at 28℃ and 120r / min for 12-24h, centrifuge them at 8000r / min for 10min, discard the supernatant, and resuspend them in sterile distilled water for 3 times. 600 The value was adjusted to 1.0, and 5% of the inoculum was inoculated into the screening medium containing 100 mg / L chloramphenicol, and cultured at 28°C and 150 r / min for 48 h.

[0040] The composition of beef extract peptone liquid culture medium is: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, distilled water 1000mL, pH 7-8, sterilization at 121℃ for 20min.

[0041] The composition of the screening medium is the same as that of step 1).

[0042] 3) Determination of chloramphenicol concentration

[0043] The bacterial liquid in the screening culture medium in step 2) was collected at 48 hours, centrifuged at 8000 rpm for 10 min, an appropriate amount of supernatant was filtered with a 0.22 μm filter membrane, and the chloramphenicol concentration in the supernatant was determined by high performance liquid chromatography: C18 chromatographic column, column temperature 30°C, mobile phase was a mixture of methanol and water in a volume ratio of 65:35, flow rate was 1 mL / min, injection volume was 10 μL, and detection wavelength was 278 nm.

[0044] Under the above conditions, the residual amount of chloramphenicol in the supernatant was measured, and the chloramphenicol degradation rate was calculated. The strain 41012 with the highest chloramphenicol degradation rate was selected, and its degradation rate for 100 mg / L chloramphenicol was 78.96%.

[0045] 4) Morphological characteristics identification

[0046] The colonies of strain 41012 are round, yellow, and moist ( Figure 1 ), the cells are rod-shaped ( Figure 2 ), Gram stain negative.

[0047] 5) Phylogenetic analysis of bacteria

[0048] For the strain 41012 with the best degradation effect obtained by repeated screening, its DNA template was prepared by alkaline lysis method, and its 16S rRNA gene was amplified by PCR using bacterial universal primers 1492R (5′-CTACGGCTACCTTGTTACGA-3′, SEQ ID NO.1) and 27F (5′-GAGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.2). The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the gene sequence length of 16S rRNA of strain 41012 was obtained to be 1410 bp (the specific sequence is as follows).

[0049]

[0050] The obtained 16S rRNA gene sequence was subjected to online Blast comparative analysis in the nucleotide standard library and EzBioCloud database. The results showed that strain 41012 belonged to the genus Chryseobacterium, so it was named Chryseobacterium sp.41012, and its phylogenetic tree was constructed ( Figure 3 ).

[0051] Example 2

[0052] Preservation of Highly Efficient Chloramphenicol Degrading Bacteria Chryseobacterium sp.41012

[0053] The strain Chryseobacterium sp.41012, whose classification name is Chryseobacterium sp., was deposited in the China Center for Type Culture Collection on November 26, 2024, with the deposit number CCTCC NO:M20242642, and the deposit address is Wuhan University, Wuhan, China.

[0054] Example 3

[0055] Preparation of highly efficient chloramphenicol-degrading bacteria Chryseobacterium sp.41012

[0056] 1) Inoculate fresh Chryseobacterium sp.41012 into sterilized beef extract peptone liquid medium and shake culture at 150r / min for 12-24h, culture temperature at 30℃, liquid volume 100 / 250mL. The beef extract peptone liquid medium is composed of: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, distilled water 1000mL, pH 7-8, sterilized at 121℃ for 20min.

[0057] 2) After the culture and fermentation are completed, the culture solution is bottled as a bacterial agent.

[0058] Example 4

[0059] Optimization of the optimal degradation efficiency of highly efficient chloramphenicol-degrading bacteria Chryseobacterium sp.41012

[0060] 1) Effects of different carbon sources on the degradation of chloramphenicol by strain Chryseobacterium sp.41012

[0061] The microbial agent prepared in Example 3 was inoculated at a 5% inoculum (w / w) into a screening culture medium with a chloramphenicol concentration of 100 mg / L and containing 1.5 g / L of different carbon sources (sodium acetate, glucose, sucrose, corn starch, wheat flour, methanol), and cultured at 150 r / min and 28°C for 48 h. The chloramphenicol content in the supernatant was determined by high performance liquid chromatography.

[0062] like Figure 4 As shown, at 48 h, when glucose and sucrose were used as carbon sources, the degradation rate of 100 mg / L chloramphenicol by the microbial agent was relatively high, at about 80%, but glucose was more economical as a carbon source.

[0063] 2) Effects of different glucose addition amounts on the degradation of chloramphenicol by strain Chryseobacterium sp.41012

[0064] The microbial agent prepared in Example 3 was inoculated at a 5% inoculum (w / w) into a screening culture medium with a chloramphenicol concentration of 100 mg / L and different glucose addition amounts (0.5, 1, 1.5, 2, 2.5, 3, 4, 5 g / L), and cultured in a shaker at 150 r / min and 28°C. The chloramphenicol content in the supernatant was determined by high performance liquid chromatography at 48 h.

[0065] like Figure 5 As shown, at 48h, when the amount of glucose added was 3g / L, the degradation rate of the microbial agent for 100mg / chloramphenicol was as high as 96.68%.

[0066] 3) Effects of different chloramphenicol concentrations on the degradation of chloramphenicol by strain Chryseobacterium sp.41012

[0067] The microbial agent prepared in Example 3 was inoculated at a 5% inoculum (w / w) into a screening medium containing 3 g / L glucose and different chloramphenicol concentrations (5, 10, 20, 30, 40, 70, 100, 170, 300 mg / L), and cultured in a shaker at 150 r / min and 28° C. The chloramphenicol content in the supernatant was determined by high performance liquid chromatography at 48 h.

[0068] like Figure 6 As shown, at 48 hours, the degradation rate of the microbial agent for 100 mg / L chloramphenicol has reached 96.45%.

[0069] 4) Effect of different pH on the degradation of chloramphenicol by strain Chryseobacterium sp.41012

[0070] The microbial agent prepared in Example 3 was inoculated at an inoculum size of 5% into a screening culture medium of different pH values ​​(5, 6, 7, 8, 9) containing 100 mg / L chloramphenicol, and cultured in a shaker at 150 r / min and 28° C. The chloramphenicol content in the supernatant was determined by high performance liquid chromatography after 48 h.

[0071] like Figure 7 As shown, when the pH range of the screening culture medium is 6-8, the microbial agent has a better degradation effect on chloramphenicol, reaching about 95%.

[0072] 5) Effect of different temperatures on the degradation of chloramphenicol by strain Chryseobacterium sp.41012

[0073] The microbial agent prepared in Example 3 was inoculated at an inoculum size of 5% into a screening medium containing 100 mg / L chloramphenicol, and cultured at 150 r / min in a shaker at different temperatures (15°C, 20°C, 28°C, 32°C, 37°C). The chloramphenicol content in the supernatant was determined by high performance liquid chromatography after 48 hours.

[0074] like Figure 8 As shown, when the culture temperature is 28-37°C, the degradation rate of chloramphenicol by the microbial agent is relatively high, reaching 94%-96%.

[0075] Example 5

[0076] Application of highly efficient chloramphenicol-degrading bacteria Chryseobacterium sp.41012 in industrial wastewater treatment

[0077] In this embodiment, actual wastewater from a sewage treatment plant in Jiaozuo City, Henan Province was used as the research object, containing tetracycline (12.22 ng / L), ofloxacin (2.27 ng / L), chloramphenicol (2.18 ng / L), erythromycin (0.96 ng / L) and clindamycin (0.56 ng / L), etc., chloramphenicol was added to the wastewater to construct low chloramphenicol concentration (5 mg / L) and high chloramphenicol concentration (100 mg / L) systems, and 3g / L of glucose was added as an external carbon source according to the optimal carbon source addition amount. The high-efficiency chloramphenicol degrading bacterial agent prepared in Example 3 was added at an inoculum size of 5% (V / V), and cultured at 28 ° C, 150r / min for 48 hours, and three parallels were set for each group.

[0078] The results show that ( Fig. 9 ), the bacterial agent has a significant effect on the degradation of chloramphenicol in industrial wastewater. When the chloramphenicol concentration is 5 mg / L, the removal rate is 62.06%; when the concentration rises to 100 mg / L, the removal rate reaches 86.97%. The bacterial agent of the present invention shows excellent performance in treating high-concentration chloramphenicol contaminated wastewater and has good industrial application prospects.

[0079] Example 6

[0080] Application of highly efficient chloramphenicol-degrading bacteria Chryseobacterium sp.41012 in the treatment of fish pond aquaculture wastewater and pond sludge

[0081] Wastewater and pond sludge from a fish pond in Jiaozuo City, Henan Province were collected, containing tetracycline (30.99 ng / L), chloramphenicol (8.77 ng / L), ofloxacin (7.22 ng / L), flumequine (6.88 ng / L) and enoxacin (5.91 ng / L), etc. After mixing evenly, chloramphenicol was added to the wastewater to construct low chloramphenicol concentration (5 mg / L) and high chloramphenicol concentration (100 mg / L) systems, 3 g / L glucose was added as the optimal external carbon source, and the high-efficiency chloramphenicol degradation bacterial agent prepared in Example 3 was added at an inoculum of 5% (V / V). Culture was carried out at 28°C and 150 r / min for 48 hours.

[0082] The results show that ( Fig. 9 ), the bacterial agent also has a good degradation effect on fish pond aquaculture wastewater and sludge: when the chloramphenicol concentration is 5 mg / L, the removal rate is 51.27%; when the concentration rises to 100 mg / L, the removal rate is 56.15%, which further verifies the application potential of the bacterial agent in actual fish farming wastewater treatment and provides an effective solution for the pollution control of aquaculture wastewater.

[0083] Example 7

[0084] Application of highly efficient chloramphenicol-degrading bacteria Chryseobacterium sp.41012 in pharmaceutical wastewater treatment

[0085] Actual wastewater from a pharmaceutical factory in Jiaozuo City, Henan Province was collected, containing clindamycin (649.59 ng / L), ciprofloxacin (213.89 ng / L), tetracycline (177.85 ng / L), ofloxacin (158.53 ng / L) and chloramphenicol (39.94 ng / L), etc. Chloramphenicol was added to the wastewater to construct low chloramphenicol concentration (5 mg / L) and high chloramphenicol concentration (100 mg / L) systems, 3 g / L of glucose was added as the optimal external carbon source, and the high-efficiency chloramphenicol degrading bacterial agent prepared in Example 3 was added at an inoculum amount of 5% (V / V), and cultured at 28°C and 150 r / min for 48 hours.

[0086] The results show that Fig. 9 ), the bacterial agent also has a certain chloramphenicol degradation effect on pharmaceutical factory wastewater. When the chloramphenicol concentration is 5 mg / L, the removal rate is 51.36%; when the concentration rises to 100 mg / L, the removal rate reaches 57.86%.

[0087] In summary, the chloramphenicol-degrading bacterial agent has broad application prospects in the treatment of chloramphenicol pollution in industrial wastewater, fish farming wastewater, and pharmaceutical wastewater, and provides a practical technical means to solve the problem of high-concentration antibiotic pollution.

[0088] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0089] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Chryseobacterium that degrades chloramphenicol, characterized in that: The Chryseobacterium was named 41012, and its classification name was Chryseobacterium sp. It was deposited in the China Center for Type Culture Collection on November 26, 2024, with the deposit number CCTCC NO:M 20242642, and the deposit address is Wuhan University, Wuhan, China.

2. Use of the Chryseobacterium described in claim 1 in degrading chloramphenicol.

3. Use of the Chryseobacterium described in claim 1 in preparing a bacterial agent for degrading chloramphenicol.

4. Use of the Chryseobacterium described in claim 1 in treating chloramphenicol-contaminated wastewater.

5. The use according to claim 4, characterized in that The wastewater is industrial wastewater, aquaculture wastewater, and pharmaceutical wastewater.

6. A chloramphenicol-degrading bacterial agent, characterized in that: The invention comprises the Chryseobacterium described in claim 1.

7. The bacterial agent according to claim 6, characterized in that The preparation method is as follows: The Chryseobacterium spp. is cultured in a beef extract peptone liquid culture medium.

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