A bacterial strain capable of degrading sulfadiazine and its combined application with ryegrass

Abstract

The present invention belongs to the field of biodegradation technology, and specifically relates to a bacterial strain capable of degrading sulfadiazine and its combined application with ryegrass. The present invention discloses sulfadiazine-degrading bacteria M9 and H1, the preservation number of M9 is: CGMCC NO.30302; the preservation number of H1 is: CGMCC NO.30301. The present invention also discloses the application of sulfadiazine-degrading bacteria and ryegrass in degrading sulfadiazine. The present invention combines sulfadiazine-degrading bacteria with ryegrass to make it have efficient sulfadiazine degradation ability, and the degrading bacteria can promote the growth of contaminated ryegrass. The results of the embodiment show that the strain has the ability to degrade sulfadiazine in the soil, and ryegrass can enhance the degradation effect by interacting with the strain.

Description

Technical Field

[0001] The invention belongs to the technical field of biodegradation, and particularly relates to a bacterial strain capable of degrading sulfadiazine and a combined application of the bacterial strain and ryegrass. Background Art

[0002] Sulfadiazine is a broad-spectrum antimicrobial drug widely used in animal husbandry and aquaculture. Due to its widespread use, sulfadiazine has caused damage to the ecological environment. The existing methods for treating sulfadiazine include the following: (1) Photocatalytic degradation: using specific catalysts and ultraviolet radiation to degrade sulfadiazine. (2) Advanced oxidation degradation: using advanced oxidation technology (such as ozone oxidation, high-pressure ultraviolet irradiation and hydrogen peroxide oxidation, etc.) to degrade sulfadiazine and convert it into non-toxic or low-toxic compounds. (3) Plant absorption: using plants with the ability to absorb sulfadiazine to absorb sulfadiazine through their roots and convert it into non-toxic or low-toxic compounds. (4) Biofiltration: using microorganisms to degrade sulfadiazine in filter beds or bioreactors, and purifying water bodies through biofiltration systems. (5) Microbial degradation: using microorganisms with degradation ability to decompose sulfadiazine into harmless substances through metabolism.

[0003] The existing methods for treating sulfadiazine still have many defects. During the physical and chemical degradation of sulfadiazine, degradation products are formed. These products have different toxicities and environmental impacts, which increases the complexity of monitoring and treatment. In addition, physical and chemical degradation methods usually require high energy consumption and expensive equipment, which is very costly. Among biodegradation, plant degradation has certain limitations. Different plants have different degradation capabilities and adaptability for sulfadiazine. In addition, some pollutants may be absorbed by plants but not metabolized, thereby causing damage to the plants themselves. Microbial degradation also has certain limitations. The efficiency of microbial degradation is affected by a series of conditions, including temperature, pH value, nutrients, etc.; and microbial degradation requires a large number of microorganisms to deal with large-scale antibiotic pollution. This involves a large amount of culture medium, oxygen supply and other conditions for maintaining the microbial flora, which increases the treatment cost and operational complexity.

[0004] Therefore, how to degrade sulfadiazine efficiently and at low cost is an urgent problem to be solved in this field. Summary of the Invention

[0005] In order to solve the above problems of the prior art, the present invention provides a strain that can efficiently degrade sulfadiazine, and an application of the strain in the combined treatment of sulfadiazine with ryegrass.

[0006] The technical solutions provided by the present invention are as follows:

[0007] The present invention provides a sulfadiazine-degrading bacterium, wherein the sulfadiazine-degrading bacterium is M9 or H1.

[0008] The deposit number of M9 is: CGMCC NO.30302; the deposit number of H1 is: CGMCC NO.30301.

[0009] The classification names of the above strains are: Acinetobacter sp.; Enterobacter sp.

[0010] The name of the depository of the above-mentioned strain is: China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit date: April 10, 2024.

[0011] The present invention also provides application of the strain in degrading sulfadiazine.

[0012] The present invention also provides the use of sulfadiazine-degrading bacteria and ryegrass in degrading sulfadiazine.

[0013] The present invention also provides a product for degrading sulfadiazine. The product comprises sulfadiazine-degrading bacteria M9 or H1. The preservation number of M9 is CGMCC NO.30302; the preservation number of H1 is CGMCC NO.30301.

[0014] The present invention also provides a method for degrading sulfadiazine in soil, which comprises planting ryegrass in sulfadiazine-contaminated soil and inoculating sulfadiazine-degrading bacteria, wherein the degrading bacteria comprises strain M9 or H1.

[0015] Preferably, the sulfadiazine-degrading bacteria are inoculated into the rhizosphere soil of ryegrass.

[0016] Preferably, the inoculation method of sulfadiazine-degrading bacteria is: adding the activated sulfadiazine-degrading bacteria suspension into the soil.

[0017] Preferably, the OD value of the suspension of sulfadiazine-degrading bacteria M9 is 0.64.

[0018] Preferably, the OD value of the suspension of sulfadiazine-degrading bacteria H1 is 0.68.

[0019] Preferably, the inoculation period of sulfadiazine-degrading bacteria is 7 days.

[0020] Beneficial effects:

[0021] The sulfadiazine-degrading bacteria disclosed in the present invention can degrade sulfadiazine independently and has the characteristics of high efficiency, selectivity, environmental friendliness and the like.

[0022] The invention combines sulfadiazine-degrading bacteria with ryegrass, so that the bacteria have high-efficiency sulfadiazine-degrading ability, and the degrading bacteria can promote the growth of contaminated ryegrass.

[0023] The present invention combines microorganisms with plants to overcome the limitations of plants alone in degrading antibiotics, leveraging their complementary strengths to enhance the effectiveness and feasibility of antibiotic degradation. Plants can modify the rhizosphere environment through compounds released from their roots, promoting the growth of beneficial microorganisms and building microbial communities conducive to degradation. Microorganisms can utilize nutrients secreted by plant roots to proliferate and degrade pollutants, thereby alleviating the damage pollutants cause to plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is the Gram staining result of M9 bacteria.

[0026] Figure 2 This is the Gram staining result of H1 bacteria.

[0027] Figure 3 This is a growth and development tree for bacterial strains.

[0028] Figure 4 This is the growth status of ryegrass in each group after 42 days.

[0029] Figure 5 The ryegrass shoot length and root length of each group after 42 days are shown, including the control group (CK-50), the M9 bacteria treatment group (M9-50), and the H1 bacteria treatment group (H1-50).

[0030] Figure 6 The degradation efficiency of sulfadiazine in the soil of each group after 42 days is as follows: control group (CK-50), M9 bacteria treatment group (M9-50), H1 bacteria treatment group (H1-50), M9 bacteria treatment group (M9-50S), and H1 bacteria treatment group (H1-50S).

[0031] Figure 7 is the leaf moisture content.

[0032] Figure 8 Chlorophyll content

[0033] Figure 9 Soil sucrase activity

[0034] Figure 10 Soil catalase activity DETAILED DESCRIPTION

[0035] The present invention provides two sulfadiazine-degrading bacteria:

[0036] The biological deposit information of strain M9 is: deposited with the China General Microbial Culture Collection, and the deposit date is April 10, 2024. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, abbreviation: CGMCC, biological deposit number: CGMCC NO. 30302, taxonomic name: Acinetobacter sp.

[0037] The biological deposit information of strain H1 is: deposited with the China General Microbial Culture Collection, and the deposit date is April 10, 2024. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, abbreviation: CGMCC, biological deposit number: CGMCC NO. 30301, taxonomic name: Enterobacter sp.

[0038] The present invention applies the above-mentioned strain to degrade sulfadiazine.

[0039] The present invention also provides a sulfadiazine-degraded product comprising the sulfadiazine.

[0040] The present invention also provides the use of sulfadiazine-degrading bacteria and ryegrass in degrading sulfadiazine.

[0041] The invention provides a method for degrading sulfadiazine in soil. The method comprises the following steps: planting ryegrass in sulfadiazine-contaminated soil and inoculating sulfadiazine-degrading bacteria.

[0042] In the present invention, the inoculation location of the sulfadiazine-degrading bacteria is not particularly limited, but is preferably in the rhizosphere soil of ryegrass.

[0043] In the present invention, the inoculation method of the sulfadiazine-degrading bacteria is not particularly limited, and preferably, the activated sulfadiazine-degrading bacteria suspension is added to the soil. The OD value of the sulfadiazine-degrading bacteria suspension is preferably 0.64 (M9 bacteria) and 0.68 (H1 bacteria). The inoculation period can be changed according to the soil conditions and is preferably 7 days.

[0044] In the present invention, strain M9 or H1 can be used to increase the water content and chlorophyll content of ryegrass leaves.

[0045] In the present invention, the strain M9 or H1 is inoculated into the rhizosphere soil of ryegrass and can be used to improve the sucrase activity and catalase activity in the soil.

[0046] The OD value in the present invention is OD 600 value.

[0047] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art.

[0049] Example 1

[0050] Preparation of sulfadiazine-degrading bacteria

[0051] Two strains were screened out from sulfadiazine-contaminated soil that could grow using sulfadiazine as the sole carbon source and named M9 and H1.

[0052] Gram staining was used to identify the two strains, and it was observed that both strains were Gram-negative bacteria. Figure 1 As shown, the staining results of strain H1 are as follows Figure 2 shown.

[0053] The selected strains were identified by physiological and biochemical methods and 16S rRNA molecular identification. M9 belonged to the genus Acinetobacter and H1 to the genus Enterobacter. Figure 3 shown.

[0054] Example 2

[0055] Efficiency of the strain in degrading sulfadiazine in soil

[0056] Activation of the strain: The strain obtained in Example 1 was activated to obtain a bacterial suspension with an OD value of 0.64 (M9 strain) and 0.68 (H1 strain). Activation was performed by dissolving 25.0 g of broth powder in 1000 mL of ultrapure water. The bacterial suspension was inoculated at a 1% concentration and cultured overnight at 37°C on a shaker. LB medium formulation (g / L): Tryptone 10.0 g; Yeast extract 5.0 g; Sodium chloride 10.0 g. pH 6.8-7.2.

[0057] Soil was contaminated with sulfadiazine to a concentration of 50 mg / kg. After seven days of stabilization, 30 mL of activated sulfadiazine-degrading bacteria were inoculated. Repeated inoculations of 30 mL of the sulfadiazine-degrading bacteria suspension were repeated every seven days for a total of 42 days. Following treatment, the residual sulfadiazine in the standard and soil samples was determined using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The degradation efficiency of each group was calculated.

[0058] According to the type of inoculated strain, the cells were divided into two groups: the M9 treatment group (M9-50S) and the H1 treatment group (H1-50S).

[0059] The degradation efficiencies of sulfadiazine in soil by the M9-50S and H1-50S groups were 27.19% and 43.39% respectively (see Table 2).

[0060] Example 3

[0061] Degradation of sulfadiazine by sulfadiazine-degrading bacteria and ryegrass

[0062] Seed treatment: Ryegrass seeds were sterilized with 70% ethanol for 5 min, 3% H2O2 for 10 min, and washed with deionized water five times.

[0063] Activation of the strain: The strain obtained in Example 1 was activated to obtain a bacterial suspension with an OD value of 0.64 (M9 strain) and 0.68 (H1 strain). Activation was performed by dissolving 25.0 g of LB liquid medium in 1000 mL of ultrapure water. The suspension was inoculated with 1% of the bacterial suspension and cultured overnight at 37°C on a shaker.

[0064] Soil was contaminated with sulfadiazine to a concentration of 50 mg / kg. After seven days of soil stabilization, seeds were evenly sown and the pots were placed in a greenhouse. After ryegrass seeds germinated, 30 mL of activated sulfadiazine-degrading bacteria were inoculated into the rhizosphere soil. During the experiment, 30 mL of the sulfadiazine-degrading bacteria suspension was repeated at 7-day intervals for a total of 42 days. Shoot and root lengths were measured in each group. After treatment, sulfadiazine residues in the standard and soil samples were determined using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The sulfadiazine degradation efficiency of each group was calculated.

[0065] According to the different inoculated strains, the animals were divided into three groups: the M9 treatment group (M9-50), the H1 treatment group (H1-50), and the control group (CK-50). The control group was not inoculated with bacteria and the treatment conditions were the same as the other two groups.

[0066] After 42 days, the growth of ryegrass in each group was as follows: Figure 4 As shown, the bud length and root length of each group are as follows Figure 5 As shown, compared with the control group (CK-50), the shoot lengths of the M9-inoculated group (M9-50) and the H1-inoculated group (H1-50) increased by 34.16% and 63.12%, respectively, and the root lengths increased by 118.71% and 162.58%, respectively. The shoot and root lengths of the ryegrass in each group are shown in Table 1.

[0067] Table 1 Shoot length and root length of each group of ryegrass

[0068]

[0069] The degradation efficiency of each group on sulfadiazine in soil after 42 days of treatment is as follows Figure 6 As shown, the degradation rates of the control group (50), the group inoculated with M9 bacteria (M9-50) and the treatment group inoculated with H1 bacteria (H1-50) were 73.73%, 92.15% and 95.88% respectively. The degradation rates of sulfadiazine in soil by each group in this example and example 1 are shown in Table 2.

[0070] Table 2 Degradation rate of sulfadiazine in soil by each group in this example and example 2

[0071]

[0072] After 42 days of treatment, the improved physiological indices of plants and soil-related indices of the control group (50), the group inoculated with M9 bacteria (M9-50) and the group inoculated with H1 bacteria (H1-50) were measured.

[0073] Leaf moisture content: Figure 7 As shown in the figure, compared with the control group, the water content of ryegrass leaves inoculated with M9 and H1 bacteria was significantly increased (P<0.05).

[0074] Chlorophyll content: Figure 8 As shown in the figure, compared with the control group, the chlorophyll content of ryegrass inoculated with M9 and H1 bacteria was significantly increased (P<0.05).

[0075] Soil sucrase activity: Figure 9 As shown in the figure, compared with the control group, the sucrase activity in the soil was significantly increased after inoculation with M9 or H1 bacteria.

[0076] Soil catalase activity: Figure 10 As shown in the figure, compared with the control group, the catalase activity in the soil was significantly increased after inoculation with M9 bacteria or H1 bacteria.

[0077] Inoculation of the M9 and H1 strains into the rhizosphere soil of ryegrass contaminated with sulfadiazine promoted ryegrass growth, with the H1 strain having a more significant effect. The inoculated strains, through interaction with the ryegrass, accelerated the degradation of sulfadiazine and provided suitable environmental conditions, thereby promoting ryegrass growth. Specifically, the M9 and H1 strains increased ryegrass leaf moisture content and chlorophyll content. Furthermore, the interaction between the inoculated strains and the ryegrass increased the activity of sucrase and catalase in the soil.

[0078] From the above examples, it can be seen that both strains M9 and H1 have the ability to degrade sulfadiazine in the soil, and the H1 strain has a higher ability to degrade sulfadiazine; after planting ryegrass, the degrading bacteria and ryegrass showed higher efficiency in degrading sulfadiazine, and the combined degradation ability of the two was significantly better than that of using the strain alone, which indicates that ryegrass can enhance the degradation effect by interacting with the strain.

[0079] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A use of a product for degrading sulfadiazine in soil for increasing the activity of sucrase and catalase in soil contaminated by sulfadiazine, characterized in that: The product comprises sulfadiazine-degrading bacteria and ryegrass; the sulfadiazine-degrading bacteria is M9 or H1, the preservation number of M9 is: CGMCC NO.30302; the preservation number of H1 is: CGMCC NO.30301; The M9 belongs to the genus Acinetobacter, and H1 belongs to the genus Enterobacter; The sulfadiazine-degrading bacteria increase the water content and chlorophyll content of the leaves of the ryegrass.

2. A method for degrading sulfadiazine in soil and increasing the activity of sucrase and catalase in the soil, characterized in that: The method comprises sowing ryegrass seeds in sulfadiazine-contaminated soil, and inoculating sulfadiazine-degrading bacteria after the ryegrass seeds germinate. The sulfadiazine-degrading bacteria include the sulfadiazine-degrading bacteria according to claim 1.

3. The method according to claim 2, characterized in that The sulfadiazine-degrading bacteria are inoculated into the rhizosphere soil of ryegrass.

4. The method according to claim 3, characterized in that The inoculation method of the sulfadiazine-degrading bacteria is: adding the activated sulfadiazine-degrading bacteria suspension into the soil.

5. The method according to claim 4, characterized in that The OD value of the suspension of the sulfadiazine-degrading bacteria M9 was 0.

64.

6. The method according to claim 4, characterized in that The OD value of the suspension of the sulfadiazine-degrading bacteria H1 was 0.

68.

7. The method according to claim 5 or 6, characterized in that The inoculation period of the sulfadiazine-degrading bacteria is 7 days.

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