Aminoglycoside antibiotic degrading bacterial agent as well as preparation method and application thereof

Abstract

The invention relates to the field of antibiotic degradation, in particular to an aminoglycoside antibiotic degradation microbial inoculum as well as a preparation method and application thereof. The aminoglycoside antibiotic degrading bacterial agent is prepared from the following raw materials: a seed solution of Chryseobacterium (Chryseobacterium sp.) (Chryseobacterium Zn-C) and iron lanthanum modified hydrothermal carbon. The preparation method comprises the following steps: (1) pretreating straws by using a ferric chloride and lanthanum chloride composite solution, and carrying out hydrothermal carbonization; (2) preparing a Chryseobacterium (Chryseobacterium sp.) (Chryseobacterium Zn-C seed solution, and preparing a seed solution of the Chryseobacterium (Chryseobacterium sp.) (Chryseobacterium Zn-C); and (3) compounding the iron-lanthanum modified hydrothermal carbon with the seed solution to form the aminoglycoside antibiotic degrading microbial inoculum. The microbial agent is put into a water body, soil or compost polluted by the aminoglycoside antibiotics to degrade the aminoglycoside antibiotics so as to achieve the pollution remediation effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibiotic degradation, in particular to an aminoglycoside antibiotic degrading microbial agent, a preparation method and application thereof. BACKGROUND

[0002] In the process of livestock breeding, the rational use of antibiotic additives can effectively prevent and treat animal infectious diseases and promote the growth of livestock.

[0003] There are thousands of known antibiotic species, among which six classes of macrolides, quinolones, beta-lactams, sulfonamides, aminoglycosides and tetracyclines are most commonly used. However, some antibiotics are stable in nature and difficult to degrade naturally in the environment. When they accumulate to a certain concentration, they can seriously damage the activity and community structure of soil and water microorganisms, and have a negative impact on the ecological environment.

[0004] During the composting process, the degradation of antibiotics is affected by multiple factors such as physicochemical properties, composting conditions, organic matter degradation and microbial action, and there are large differences in the degradation effect of different types of antibiotics. Microbial degradation method has important application value in the treatment of antibiotic residues in livestock breeding waste because it is an economical and effective means to decompose antibiotics into low-toxic or non-toxic small molecular products due to its simple and controllable conditions and low cost. For example, patent CN201610177887.0 discloses an aminoglycoside antibiotic sewage bacteria FN-A3, which is cultured in 1000mg / L gentamicin sewage. After detection, the degradation rate of gentamicin in the water body is 22.7% in 24 hours, 56.40% in 36 hours, 84.40% in 48 hours, and 99% in 72 hours.

[0005] However, the current biological degradation technology relies solely on bacterial strains, which may lead to a decrease in degradation efficiency due to environmental conditions (such as pH and temperature fluctuations), and has low stability and the problem of secondary pollution caused by free bacterial strains. Therefore, it has good application prospects to develop high-efficiency microbial immobilized degradation materials to solve the problems of unstable degradation effect and engineering application. SUMMARY

[0006] The technical problem to be solved by the present application is to provide an aminoglycoside antibiotic degrading microbial agent, a preparation method and application thereof.

[0007] The present application is implemented as follows:

[0008] The application first provides an aminoglycoside antibiotic degrading bacterial agent, raw materials for preparation of the aminoglycoside antibiotic degrading bacterial agent including seed liquid of Chryseobacterium sp. Chryseobacterium Zn-C and iron-lanthanum modified hydrothermal carbon, the Chryseobacterium sp. Chryseobacterium Zn-C is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address of the preservation is No. 1, Yitianxi Li, Chaoyang District, Beijing, the date of the preservation is April 12, 2019, and the preservation number is CGMCC NO. 17564.

[0009] The preparation method of the iron-lanthanum modified hydrothermal carbon includes the following steps:

[0010] (a) crushing crop straw;

[0011] (b) preparing a compound solution of 10-15 mg / L of ferric chloride and 5-8 mg / L of lanthanum chloride, adding the compound solution into the crushed straw and immersing for more than 12 hours, and stirring for three times during the immersing;

[0012] (c) transferring the straw mixture obtained in step b to a hydrothermal reaction kettle for hydrothermal carbonization, and after cooling, performing solid-liquid separation to obtain the iron-lanthanum modified hydrothermal carbon material.

[0013] Further, the crushing in step a is crushing the straw to a particle size of less than 2 mm.

[0014] Further, the hydrothermal carbonization in step c is performed at a temperature of 300-320 DEG C for 4-6 hours.

[0015] Further, the mass concentration of the iron-lanthanum modified hydrothermal carbon in the raw materials is 10%-15%, the seed liquid accounts for 5%-10% (volume ratio), and the rest is sterile water; after the mixed solution obtained by mixing the raw materials is left overnight at room temperature, solid-liquid separation and freeze-drying are performed to obtain the aminoglycoside antibiotic degrading bacterial agent.

[0016] The application also provides a preparation method of the aminoglycoside antibiotic degrading bacterial agent, specifically including the following steps:

[0017] (1) crushing crop straw to a particle size of less than 2 mm;

[0018] (2) preparing a compound solution of 10-15 mg / L of ferric chloride and 5-8 mg / L of lanthanum chloride, adding the compound solution into the crushed straw at a solid-liquid ratio of 1:4-1:6 (volume ratio) and immersing for more than 12 hours, and stirring for three times during the immersing;

[0019] (3) The obtained straw mixture was transferred to a hydrothermal reactor and hydrothermally carbonized at 300℃-320℃ for 4-6 hours; after water cooling, solid-liquid separation was performed to obtain iron-lanthanum modified hydrothermal carbon.

[0020] (4) Preparation of Chryseobacterium Zn-C seed culture: Chryseobacterium Zn-C strain was incubated in LB liquid medium at 30℃ for 150 r·min -1 Incubate for 12-24 hours under the specified conditions, then at 12000 r·min -1 Centrifuge for 5 min to collect bacterial cells. Wash the bacterial cells three times with sterile water rinse solution and resuspend them in an equal volume of sterile water to prepare seed culture. The effective viable count in the seed culture is >10. 9 cfu / ml.

[0021] (5) Preparation of aminoglycoside antibiotic degrading bacterial agent: The iron-lanthanum modified hydrothermal carbon and the...

[0022] Chryseobacterium Zn-C seed liquid is mixed, and the mass concentration of the iron-lanthanum modified hydrothermal carbon in the mixed solution is 10%-15%, the proportion of Chryseobacterium Zn-C seed liquid is 5%-10% (volume ratio), and the remainder is sterile water; after the prepared mixed solution is left to stand overnight at room temperature, solid-liquid separation is performed and freeze-dried to obtain an aminoglycoside antibiotic degrading bacterial agent.

[0023] The present invention also provides the application of the aminoglycoside antibiotic degrading agent in aminoglycoside antibiotic contaminants.

[0024] Furthermore, the aminoglycoside antibiotic contaminants include water, soil, or compost.

[0025] Compared with existing technologies, the product of this invention has advantages such as faster degradation, wider adaptability, more environmental friendliness, lower production costs, and greater ease of use. Specifically, these advantages are reflected in:

[0026] (1) It has high degradation efficiency, which is more than 20% higher than the removal efficiency of single strains. Furthermore, the iron-lanthanum modified hydrothermal carbon can simultaneously adsorb and catalytically decompose antibiotic molecules, achieving "adsorption-biodegradation" coupling and avoiding secondary release.

[0027] (2) The degradation agent has high stability and wide applicability. It can be used in sewage reactors, or directly applied to acidic soil or high-temperature composting without additional process modification. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Figure 1A plot of the degradation rate of gentamicin in soil samples as a function of incubation time.

[0030] Figure 2 A plot of the degradation rate of gentamicin in soil samples as a function of incubation time.

[0031] Figure 3 A plot of the degradation rate of gentamicin in soil samples as a function of incubation time. DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be described clearly and completely in combination with the drawings and specific embodiments. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market. Among them, the patent CN201911184639.9 A kind of gold yellow bacillus with heavy metal resistance and its application discloses gold yellow bacillus (Chryseobacterium sp.) Chryseobacterium Zn-C.

[0033] Example 1

[0034] A preparation method of an aminoglycoside antibiotic degrading microbial agent, specifically comprising the following steps:

[0035] (1) The crop straw is crushed to a particle size of <2 mm;

[0036] (2) A composite solution of 10 mg / L of ferric chloride and 5 mg / L of lanthanum chloride is prepared, and is added to the crushed straw at a solid-liquid ratio of 1:4 (volume ratio) and soaked for 12 h, with stirring for 3 times during the period;

[0037] (3) The obtained straw mixture is transferred to a hydrothermal reaction kettle, and is hydrothermally carbonized at 300℃ for 6h; after water cooling, solid-liquid separation is performed to obtain iron-lanthanum modified hydrothermal carbon;

[0038] (4) Preparation of Chryseobacterium Zn-C seed liquid: Chryseobacterium Zn-C strain is cultured in LB liquid medium at 30℃ and 150r·min-1 for 12-24h, then centrifuged at 12000r·min-1 for 5min to collect the bacterial bodies, which are washed with sterile water for 3 times and then resuspended in an equal volume of sterile water to prepare the seed liquid, and the effective viable bacterial count in the seed liquid is >10 9 cfu / ml.

[0039] (5) Preparation of an aminoglycoside antibiotic degrading microbial agent: the iron-lanthanum modified hydrothermal carbon and the

[0040] Chryseobacterium Zn-C seed liquid is mixed uniformly, and in the mixed solution, the mass concentration of the iron-lanthanum modified hydrothermal carbon is 10%, the Chryseobacterium Zn-C seed liquid accounts for 5% (volume ratio), and the rest is sterile water; after the prepared mixed solution is left at room temperature overnight, solid-liquid separation and freeze-drying are performed, to obtain an aminoglycoside antibiotic degrading agent.

[0041] Application Example 1

[0042] The formula of the inorganic salt basic medium is K2HPO41.6 g, MgSO40.2 g, CaCl20.025 g, FeCl30.0023 g, and distilled water 1000 mL, which is sterilized at 121 ℃ for 20 min.

[0043] The aminoglycoside antibiotic degrading agent (iron-lanthanum modified hydrothermal carbon-Chryseobacterium Zn-C composite material) of the present application is added into an inorganic salt basic medium with gentamicin or kanamycin as the only carbon source at an addition amount of 1%, and is cultured at 37 ℃, 150 r / min, and in the dark for 24 h; solution samples are collected at the 3rd, 6th, 12th, 18th, and 24th hours after addition, and the content of aminoglycoside antibiotics in the culture solution is determined by high performance liquid chromatography-evaporative light detector (HPLC-ELSD). An unseeded medium is used as a blank control, and 5 parallels are set for each experimental group. The calculation formula of the aminoglycoside antibiotic degradation rate is as follows:

[0044] Degradation rate = (C0-Ct) / C0 x 100% t

[0045] In the formula, C0 is the concentration of aminoglycoside antibiotics in the unseeded culture solution, mg / L; and Ct is the concentration of aminoglycoside antibiotics in the seeded culture solution after a certain period of culture, mg / L.

[0046] It is detected that the degradation rate of gentamicin in the solution reaches 79.56% after 6 hours of treatment, and is completely degraded after 12 hours. The degradation rate of kanamycin solution reaches 41.26% after 3 hours of culture, 81.33% after 6 hours of culture, and is completely degraded after 12 hours. The degradation rate changes with the culture time as shown in Figure 1 .

[0047] Application Example 2

[0048] 100 g of soil with an initial gentamicin content of 20 mg / kg is weighed, and then 0.1% of Chryseobacterium Zn-C seed liquid and 2% of unmodified hydrothermal carbon immobilized Chryseobacterium Zn-C corresponding to 0.1% of the mass of the soil are added to the soil, respectively. ​

[0049] Chryseobacterium Zn-C, 2% of iron lanthanum modified hydrothermal carbon immobilized Chryseobacterium Zn-C composite material, with contaminated soil without adding any material as a control group (CK), repeated 3 times. The weighing method was used to control the soil water content to be 60% of the field water capacity, and the soil was incubated at 30 DEG C for 15 days, and the soil samples were collected at 5th and 15th days of incubation to determine the gentamicin content.

[0050] It was detected that, as shown in Figure 2 after 5 days of incubation, the degradation rate of gentamicin treated by Chryseobacterium Zn-C was 80.9%, the degradation rate of gentamicin treated by unmodified hydrothermal carbon immobilized degradation bacteria was 89.7%, and the degradation rate of gentamicin treated by iron lanthanum modified hydrothermal carbon immobilized Chryseobacterium Zn-C reached 96.65%, and the gentamicin was below the detection limit after 15 days of incubation. The iron lanthanum modified hydrothermal carbon immobilized Chryseobacterium Zn-C system not only can improve the degradation efficiency, but also can realize the rapid and complete removal of gentamicin within 15 days, which indicates that the iron lanthanum modified hydrothermal carbon as a carrier can significantly improve the catalytic activity and stability of the bacteria, and has the potential for engineering application.

[0051] Application Example 3

[0052] The chicken manure and the fungus residue were mixed according to a certain proportion, so that the carbon-nitrogen ratio of the mixed raw materials was 30:1. The composting test was carried out by adding kanamycin externally, and the theoretical content of kanamycin in the composting raw materials was 100 mg / kg. In the test, 5% (weight ratio) of hydrothermal carbon and iron lanthanum modified hydrothermal carbon immobilized Chryseobacterium Zn-C material were added to the mixture, and a control group (CK) was set at the same time. The water content was adjusted to 60%, and the composting samples were collected at 5th and 15th days of composting to determine the content of kanamycin in the composting.

[0053] It was detected that, as shown in Figure 3 at 5th day of composting, the degradation rate of kanamycin treated by single hydrothermal carbon was 84.5%, the degradation rate of kanamycin treated by iron lanthanum modified hydrothermal carbon immobilized Chryseobacterium Zn-C reached 92.4%, and the degradation rate of kanamycin reached 100% after 15 days of incubation, which indicates that the degradation bacteria agent of the present application has the adsorption-catalysis function of iron lanthanum modified hydrothermal carbon and

[0054] The biodegradation activity of Chryseobacterium Zn-C degradation bacteria can quickly adsorb and continuously decompose antibiotics in the high-temperature composting stage, block the environmental risk of antibiotics entering the soil-plant system with composting products, and provide an efficient and economical repair path for the safe resourceization of livestock and poultry manure.

[0055] While the foregoing describes specific embodiments of the application, one of ordinary skill in the art will further appreciate that the specific exemplary embodiments described are meant to be illustrative only and are not intended to limit the scope of the application. Changes, modifications, and equivalents which would occur to one skilled in the art upon a reading of the foregoing description are meant to be encompassed within the scope of the application.

Claims

1. An aminoglycoside antibiotic degrading agent, characterized in that: The raw materials for preparation include seed culture of Chryseobacterium sp. Chryseobacterium Zn-C and iron-lanthanum modified hydrothermal carbon. The Chryseobacterium sp. Chryseobacterium Zn-C is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 12, 2019, with accession number CGMCCNO.17564.

2. The aminoglycoside antibiotic degrading agent according to claim 1, characterized in that: The preparation method of the iron-lanthanum modified hydrothermal carbon includes the following steps: (a) Crushing crop straw; (b) Prepare a composite solution of 10-15 mg / L ferric chloride and 5-8 mg / L lanthanum chloride, add it to the crushed straw and soak for more than 12 hours, stirring 3 times during the process; (c) The straw mixture obtained in step b is transferred to a hydrothermal reactor for hydrothermal carbonization; after cooling, solid-liquid separation is performed to obtain iron-lanthanum modified hydrothermal carbon material.

3. The aminoglycoside antibiotic degrading bacterial agent according to claim 2, characterized in that: The crushing mentioned in step a refers to crushing the straw to a particle size of less than 2 mm.

4. The aminoglycoside antibiotic degrading agent according to claim 2, characterized in that: The hydrothermal carbonization conditions described in step c are hydrothermal carbonization at 300℃-320℃ for 4-6 hours.

5. The aminoglycoside antibiotic degrading bacterial agent according to claim 1, characterized in that: The mass concentration of the iron-lanthanum modified hydrothermal carbon in the raw materials is 10%-15%, the volume ratio of the seed liquid is 5%-10%, and the remainder is sterile water. After the mixed solution obtained by mixing the raw materials is left to stand overnight at room temperature, solid-liquid separation and freeze-drying are performed to obtain an aminoglycoside antibiotic degrading bacterial agent.

6. A method for preparing an aminoglycoside antibiotic degrading bacterial agent as described in any one of claims 1-5, characterized in that: Specifically, the steps include the following: (1) Crush the crop straw to a particle size of <2mm; (2) Prepare a composite solution of 10-15 mg / L ferric chloride and 5-8 mg / L lanthanum chloride, add it to the crushed straw at a solid-liquid ratio of 1:4-1:6 and soak for more than 12 hours, stirring 3 times during the process; (3) The obtained straw mixture was transferred to a hydrothermal reactor and hydrothermally carbonized at 300℃-320℃ for 4-6 hours; after water cooling, solid-liquid separation was performed to obtain iron-lanthanum modified hydrothermal carbon. (4) Preparation of Chryseobacterium sp. Zn-C seed culture: Chryseobacterium Zn-C strain was incubated in LB liquid medium at 30°C for 150 rpm. -1 Incubate for 12-24 hours under the specified conditions, then at 12000 r·min -1 Centrifuge for 5 min to collect bacterial cells. Wash the bacterial cells three times with sterile water rinse solution and resuspend them in an equal volume of sterile water to prepare seed culture. The effective viable count in the seed culture is >10. 9 cfu / ml; (5) Preparation of aminoglycoside antibiotic degrading bacterial agent: The iron-lanthanum modified hydrothermal carbon and the Chryseobacterium Zn-C seed liquid are mixed. In the mixed solution, the mass concentration of the iron-lanthanum modified hydrothermal carbon is 10%-15%, the volume ratio of the Chryseobacterium Zn-C seed liquid is 5%-10%, and the remainder is sterile water. After the prepared mixed solution is left to stand overnight at room temperature, solid-liquid separation is performed and freeze-dried to obtain the aminoglycoside antibiotic degrading bacterial agent.

7. The application of the aminoglycoside antibiotic degrading bacterial agent as described in claims 1-5 or the aminoglycoside antibiotic degrading bacterial agent prepared by the preparation method as described in claim 6 in aminoglycoside antibiotic pollutants.

8. The application according to claim 7, characterized in that: The aminoglycoside antibiotic contaminants include water, soil, or compost.

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