Marine streptomyces DCF24 and application thereof in degrading chlorphenyl compound

Abstract

The invention discloses marine streptomyces DCF24 and an application of the marine streptomyces DCF24 in degradation of chlorphenyl compounds. The marine streptomyces DCF24 is preserved in the China Center for Type Culture Collection on November 27, 2024, and the preservation number of the marine streptomyces DCF24 is CCTCC NO: M 20242649. The streptomyces marinus DCF24 is applied to degradation of chlorphenyl compounds, particularly has high capacity of degrading the non-steroidal anti-inflammatory drug diclofenac, can degrade for 7 days under the anaerobic condition, has the degradation rate of 95%, is suitable for treating pollution of the non-steroidal anti-inflammatory drug diclofenac in the environment, and can achieve the purpose of environmental remediation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of marine Streptomyces. More particularly, it relates to a marine Streptomyces DCF24 capable of degrading chlorophenyl compounds and its application. BACKGROUND

[0002] Chlorophenyl compounds refer to organic compounds with one or more chlorine atoms (Cl) substituted on the benzene ring, which are widely used in the fields of medicine, pesticide, material science, etc. Diclofenac (DCF) is a typical dichlorophenyl compound (2,6-dichloroaniline derivative). As a non-steroidal anti-inflammatory drug, diclofenac is widely used in the treatment of various inflammatory diseases, which easily leads to pollution of surface water, groundwater and even drinking water. At present, the efficiency of wastewater treatment plants in removing diclofenac is low, and it is usually discharged into the aquatic environment without sufficient treatment. Moreover, due to the low concentration and relatively high polarity of diclofenac in the environment, it is difficult to be effectively removed. In addition, when diclofenac exists in the water supply system, it may cause the expression of antibiotic resistance genes, leading to the production of drug-resistant bacteria or so-called superbugs. In order to effectively address this problem, it is urgent to strengthen the research on monitoring and removal technology of diclofenac in water environment to ensure the safety of water resources and the health of ecological environment.

[0003] There are physical, chemical, biological and advanced oxidation methods for the treatment of non-steroidal anti-inflammatory drug pollution, and the microbial method has the advantages of economy, high efficiency and complete degradation. Therefore, it is of great scientific significance and application value to use activated sludge to enrich and domesticate, and purify and separate strains capable of efficiently degrading diclofenac, and to study their characteristics for the treatment of diclofenac environmental pollution. However, there is a lack of high-efficiency strains for degrading diclofenac, which undoubtedly brings great obstacles to environmental pollution control. Chinese Patent Publication No. CN105753154A uses a sponge iron-ammonia-oxidizing microbial system to simultaneously remove anti-inflammatory drugs and ammonia nitrogen, which is too complex. So far, there is no literature report on the ability of marine Streptomyces (Streptomyces maritimus) to degrade diclofenac. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a marine Streptomyces (Streptomyces maritimus) DCF24 capable of degrading diclofenac and its application. The marine Streptomyces (Streptomyces maritimus) DCF24 has strong degradation ability for diclofenac under anaerobic conditions, and the strain is suitable for treating diclofenac pollution in the environment and can achieve the purpose of environmental remediation.

[0005] A first object of the present application is to provide a Streptomyces maritimus DCF24.

[0006] A second object of the present application is to provide an application of the Streptomyces maritimus.

[0007] A third object of the present application is to provide a preparation for degrading chlorobenzene compounds.

[0008] To achieve the above objects, the present application adopts the following technical solutions.

[0009] The present application separates a Streptomyces maritimus DCF24 with the function of degrading chlorobenzene compounds under anaerobic conditions from an aerobic reactor added with diclofenac (DCF), and the strain can effectively degrade diclofenac under a single carbon source substrate environment, and the degradation efficiency can reach 95%.

[0010] The Streptomyces maritimus DCF24 provided by the present application can effectively degrade diclofenac under a single carbon source.

[0011] The present application first determines the Streptomyces maritimus DCF24 with the function of degrading chlorobenzene compounds, and the Streptomyces maritimus DCF24 has been preserved in the China Center for Type Culture Collection on November 27, 2024, with the preservation number of CCTCC NO: M 20242649.

[0012] The application of the Streptomyces maritimus DCF24 in degrading chlorobenzene compounds. The Streptomyces maritimus is used for degradation, and the biological adsorption effect on chlorobenzene compounds is excluded.

[0013] Preferably, the degradation conditions are as follows: under anaerobic conditions, the temperature is 25-35℃, and the pH is 6.5-7.5.

[0014] Preferably, the degradation conditions are as follows: the temperature is 30.4±2℃, and the pH is 7.1±0.2.

[0015] Preferably, the initial inoculation amount of the Streptomyces maritimus DCF24 is 0.5-5.0 mg / mL.

[0016] Preferably, the initial inoculation amount of the Streptomyces maritimus DCF24 is 1-1.5 mg / mL.

[0017] A preparation for degrading chlorobenzenyl compounds, the preparation comprising the marine Streptomyces DCF24.

[0018] Preferably, the preparation is a preparation for degrading chlorobenzenyl compounds in a water body.

[0019] Preferably, the chlorobenzenyl compounds include one or two or more of diclofenac, indometacin, tolmetin, lodoxacabane.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The marine Streptomyces DCF24 provided by the present application has strong ability to degrade non-steroidal anti-inflammatory drug diclofenac, and under anaerobic conditions, the degradation rate can reach 95% after 7 days of degradation. The marine Streptomyces DCF24 provided by the present application is especially suitable for treating non-steroidal anti-inflammatory drug diclofenac pollution in the environment, and can achieve the purpose of environmental remediation.

[0022] The marine Streptomyces DCF24 has been preserved in the China Center for Type Culture Collection (CCTCC) on November 27, 2024, with a preservation number of CCTCC NO: M 20242649, and a preservation address of Wuhan University, Wuhan, Hubei, China. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A phylogenetic tree of marine Streptomyces DCF24 and other reported strains with halogenated organic matter degradation effect.

[0024] Figure 2 A single metabolic degradation effect diagram (a) and dechlorination effect diagram (b) of marine Streptomyces DCF24 on diclofenac.

[0025] Figure 3 A biological degradation of diclofenac under co-metabolic conditions and a non-biological factor control.

[0026] Figure 4 A co-metabolic degradation effect diagram of marine Streptomyces DCF24 under different initial concentrations of diclofenac.

[0027] Figure 5 A degradation effect diagram of marine Streptomyces DCF24 on different chlorobenzenyl compounds.

[0028] Figure 6Scanning electron microscope (SEM) image of Streptomyces maritimus DCF24. DETAILED DESCRIPTION

[0029] The application will be further described below in conjunction with the accompanying drawings and specific examples. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0030] Example 1 Isolation and identification of Streptomyces maritimus DCF24.

[0031] 1. Enrichment and isolation of the strain

[0032] The aerobic sludge was taken from the second water purification plant in Xinyi City, Guangdong Province, and washed according to the following steps: ① remove large particles, stand for 30 min, discard the supernatant; ② add PBS (phosphate buffer saline) solution to resuspend, vortex for 10 min, discard the supernatant. Take 20 mL of pure sludge and add 200 mL of nutrient solution (composition and content see Table 1), add diclofenac (DCF) to 1-10 mg / L. Under uniform stirring of the rotor, take 0.1 mL of bacterial suspension (diluted 10 1 ~ 10 4 times) and spread on MSM solid plate medium with diclofenac as the sole carbon source at 37°C in the dark until single pure colonies appeared on the plate. Select colonies with good growth and regular edges for multiple purification. The MSM medium contains 0.002 g / L of diclofenac, 1.5 g / L of agar powder, and other added components and contents as shown in Table 1.

[0033] Table 1: Composition and content of nutrient solution

[0034]

[0035] 2. Characteristics and identification of the strain

[0036] The marine Streptomyces grows on MSM plates, with round, light gray, rough and dry surface, jagged edges, and no halo color. The main physicochemical properties of Streptomyces maritimus DCF24 are: strain growth temperature 20-40°C, pH range 6-8, facultative anaerobic growth, positive for protease and amylase. Scanning electron microscope (SEM) image is shown in Figure 6 .

[0037] Molecular marker identification: The total DNA of the bacteria was extracted by using a bacterial DNA extraction kit, the 16S rRNA sequence of the above strain was amplified by PCR using bacterial 16S rRNA universal primers, then BLAST comparison was performed, and the phylogenetic tree was constructed by using MEGAX software to obtain the phylogenetic relationship tree of the strain Figure 1 ).

[0038] The PCR amplification conditions are as follows:

[0039] Universal primers:

[0040] 27F: AGAGTTTGATCMTGGCTCAG

[0041] 1492R: GGTTACCTTGTTACGACTT

[0042] Table 2 reaction system

[0043] Reaction components Volume (μl) 10X PCR Buffer dNTP (each 10 mM) Taq Plus DNA Polymerase (5 U / μl) 50 mM MgSO4 Total 12.5 Primer F (10 μM) 1 Primer R (10 μM) 1 Template (DNA) 1 ddH2O 9.5 Total 25

[0044] Table 3 PCR amplification procedure

[0045]

[0046] The results of BLAST comparison of the sequencing of its 16S rRNA showed that the 16S rRNA sequence of the strain had a homology of 100% with Streptomyces maritimus. Therefore, we can judge that the strain belongs to Streptomyces maritimus, and is named Streptomyces maritimus DCF24.

[0047] Example 1: Identification of the strain by 16S rRNA, the 16S rRNA sequencing result of Streptomyces maritimus DCF24 is shown as SEQ ID No. 1.

[0048] Example 2: DCF degradation test of Streptomyces maritimus DCF24 under anaerobic conditions at different pH, temperature and initial bacterial amount.

[0049] 1. Isolated and purified Streptomyces maritimus DCF24 was used to degrade diclofenac in a conical flask experiment under anaerobic conditions. The degradation rate of diclofenac was detected. Temperature (20-40℃), pH (6-8) and initial bacterial amount (0.5-1.5 mg / mL) were used as independent variables, and the degradation rate was used as dependent variable. A three-factor and three-level response surface analysis was performed, the design scheme was Box-Behnken, and the fitting model was Quadratic. The experimental data is shown in Table 4 below:

[0050] Table 4 Response surface data results

[0051]

[0052]

[0053] 2. Under the condition of using diclofenac as a single carbon source, the corresponding surface analysis model Adjusted R 2 was 0.9645, and the fitting equation was as follows:

[0054] Degradation rate (%) = +0.92 + 0.036*A + 0.039*B + 0.032*C + 0.052*AB + 0.025*AC - 0.045*BC

[0055] wherein A is pH, B is temperature (℃), and C is initial bacterial amount. Therefore, the optimal experimental conditions were selected as temperature 30.4℃, pH 7.1, and initial bacterial amount 1 mg / mL. The fitting degradation rate can reach 96% by Design Expert 11 software fitting. In actual operation, the temperature was controlled at 30℃, the pH was controlled at 7.3, and the initial bacterial amount was controlled at 1 mg / mL.

[0056] Example 3

[0057] Under the condition of single metabolism of Streptomyces maritimus DCF24 under anaerobic and optimal conditions (30℃, pH 7.3, and initial bacterial amount 1 mg / mL), the degradation experiment of diclofenac (DCF) was performed with an initial concentration of diclofenac of 2.2 mg / L. The degradation rate of diclofenac and the dechlorination condition were detected, and the results are shown in Figure 2 After 7 days of degradation, the degradation rate can reach 95%, and the content of chloride ions increases from 0 to 0.3 mg / L. It is proved that the strain not only has a certain degradation efficiency on diclofenac, but also has the ability of dechlorination.

[0058] Diclofenac degradation rate:

[0059] Example 4

[0060] Inactivation of Streptomyces maritimus DCF24 and adsorption interference after inactivation, after inactivation, the strain was used for adsorption experiment of diclofenac (initial concentration 2.2 mg / L, 1% volume fraction of methanol). The concentration of diclofenac in the inactivated experimental group did not decrease obviously, which proved that the strain completed the degradation of diclofenac through the metabolic process, and excluded the interference of non-biological factors. The results are shown in Table 1. Figure 3 .

[0061] Example 5

[0062] The initial concentration of diclofenac was set to 4 mg / L, 6 mg / L, 8 mg / L and 10 mg / L. When the mother liquor was added, methanol was added to the system to promote dissolution. The volume fraction of methanol was 1%, the initial pH was adjusted to 7, and the initial bacterial amount was 1 mg / mL. The culture was placed in a 100 mL conical flask. Under the condition of co-metabolism of different concentrations of methanol, Streptomyces maritimus DCF24 showed certain degradation capacity, as shown in Table 2. Figure 4 When the concentration rose to 10 mg / L, the degradation rate could reach 40%, which indicated that the strain could adapt to the concentration range of diclofenac of 0-10 mg / L, and was limited by the solubility of the substance, and special cases exceeding 10 mg / L were not considered.

[0063] Example 6

[0064] The separated and purified Streptomyces maritimus DCF24 was used for conical flask experiment of degradation of diclofenac, triclofan, indomethacin and tolmetin under anaerobic conditions. The initial concentration was 2.2 mg / L, and the initial bacterial amount was 1 mg / mL. The degradation rates were 95%, 53%, 44% and 10% respectively, which indicated that the strain also had certain degradation capacity for other chlorophenyl compounds. The results are shown in Table 3. Figure 5 .

[0065] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. For ordinary skilled persons in the art, on the basis of the above description and ideas, other different forms of changes or variations can also be made, which do not need to be or cannot be exhaustively listed here. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A marine Streptomyces DCF24, characterized in that, The marine Streptomyces maritimus DCF24 has been deposited with the China Center for Type Culture Collection on November 27, 2024, and the deposit number is CCTCC NO: M20242649.

2. The marine Streptomyces maritimus DCF24 of claim 1 is used for degrading chlorophenyl compounds.

3. Use according to claim 2, characterized in that, The chlorophenyl compounds include one or more of diclofenac, indometacin, tolmetin, lodoxacabane and chloroquine.

4. Use according to claim 2, characterized in that, The degradation condition is that the temperature is 25-35℃ and the pH is 6.5-7.5 under anaerobic condition.

5. Use according to claim 4, characterized in that, The degradation condition is that the temperature is 30.4±2℃ and the pH is 7.1±0.

2.

6. Use according to claim 2 or 3 or 4 or 5, characterized in that, The initial inoculum of the marine Streptomyces maritimus DCF24 is 0.5-5.0 mg / mL.

7. Use according to claim 6, characterized in that, The initial inoculum of the marine Streptomyces maritimus DCF24 is 1-1.5 mg / mL.

8. A preparation for degrading chlorophenyl compounds, characterized by, The preparation contains the marine Streptomyces maritimus DCF24 of claim 1.

9. The formulation of claim 8, wherein, The preparation is used for degrading chlorophenyl compounds in water.

10. The preparation according to claim 9, characterized in that The chlorophenyl compounds include one or more of diclofenac, indometacin, tolmetin, lodoxacabane and chloroquine.

Images