Citrobacter murlae strain dh3 and application thereof in adsorbing heavy metals and / or degrading doxycycline

Abstract

The application provides a Citrobacter murliniae strain DH3 and application thereof in adsorbing heavy metals and / or degrading doxycycline, and belongs to the technical field of environmental microorganisms. The application provides a Citrobacter murliniae strain DH3, and biological preservation has been completed. The Citrobacter murliniae strain DH3 can tolerate various heavy metals including Cu 2+ , Pb 2+ , Cr 6+ , Cd 2+ , has high removal capacity for Pb 2+ and / or Cd 2+ , and has high degradation capacity for doxycycline. Tests find that the Citrobacter murliniae strain DH3 can tolerate the compound pollution conditions of heavy metals and doxycycline, and can achieve the effect of simultaneously removing heavy metals and degrading doxycycline, thereby providing new strain resources for the treatment of aquaculture water pollution.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a strain of Citrobacter murineis DH3 and its application in adsorbing heavy metals and / or degrading doxycycline. Background Technology

[0002] Heavy metals cannot be degraded by microorganisms and exist stably in the environment primarily as precipitates, oxides, etc., adhering to particles. During remediation, it is difficult to ensure sufficient contact and reaction between the remediation agent and the heavy metals, making heavy metal pollution difficult to treat. Heavy metals easily accumulate in organisms and enter the human body through the food chain, seriously threatening human health. Doxycycline wastewater contains high levels of suspended solids (SS) and residual antibiotics, making direct biological treatment difficult. When doxycycline remains in the environmental medium, it is affected by biological or abiotic processes, causing its parent compound to change from large molecules to smaller molecules, ultimately forming H2O and CO2. Due to the extreme complexity of influencing factors in the environmental medium, various degradation pathways exist, mainly divided into two categories: abiotic and biodegradation.

[0003] Existing methods for removing heavy metals and doxycycline each have their advantages, but they also have drawbacks to varying degrees, such as high investment, high energy consumption, difficult operation, and the potential for secondary pollution. Microbial degradation is one of the safest and most efficient methods for removing heavy metals and antibiotics. It refers to the process in which microorganisms, through metabolism in a specific environment, produce enzymes and other substances that directly or indirectly alter the parent structure of antibiotics, thereby inactivating them. Consequently, more and more microorganisms are being discovered and used for environmental pollutant remediation. Some important strains, such as Bacillus and Pseudomonas, have been widely used to remove heavy metals from wastewater and soil due to their high metal-binding affinity. However, research on the microbial degradation of doxycycline is scarce. Furthermore, most studies focus on the removal of single pollutants, with limited research on screening strains that demonstrate good removal effects for both doxycycline and heavy metals. Summary of the Invention

[0004] The purpose of this invention is to provide a Citrobacter murine strain DH3 that has the dual function of adsorbing heavy metals and degrading doxycycline, and the effect is excellent.

[0005] This invention provides a strain of Citrobacter murliniae, DH3, with accession number CGMCC No. 29865.

[0006] The present invention also provides a microbial agent comprising Citrobacter murineis DH3 as described in the above technical solution.

[0007] Preferably, the OD of the Citrobacter murine strain DH3 in the bacterial agent is... 600 It ranges from 0.5 to 2.

[0008] Preferably, the microbial agent comprises the cell body of the Citrobacter murine strain DH3 or the fermentation broth of the Citrobacter murine strain DH3.

[0009] The present invention also provides the application of the Citrobacter murine strain DH3 or the bacterial agent described in the above technical solutions in one or more of the following: treatment of aquaculture wastewater, removal of heavy metals, and degradation of doxycycline.

[0010] Preferably, the treatment of aquaculture wastewater includes removing heavy metals and / or doxycycline from the aquaculture wastewater.

[0011] Preferably, the heavy metal includes Pb. 2+ and / or Cd 2+ The concentration of the heavy metal is 5–50 mg / L.

[0012] The concentration of the doxycycline is 20–200 mg / L.

[0013] This invention also provides a method for reducing heavy metal and / or doxycycline pollution in aquaculture wastewater, wherein Citrobacter murineis strain DH3 is mixed with aquaculture wastewater for culture; the preservation number of Citrobacter murineis strain DH3 is CGMCC No. 29865.

[0014] Preferably, the amount of *Citrobacter murineis* strain DH3 added is based on the bacterial solution containing *Citrobacter murineis* strain DH3, and the volume ratio of the bacterial solution of *Citrobacter murineis* strain DH3 to the aquaculture wastewater is (0.5-2):100; the OD of the bacterial solution of *Citrobacter murineis* strain DH3... 600 It ranges from 0.5 to 2.

[0015] Preferably, the heavy metal includes Pb. 2+ and / or Cd 2+ The concentration of the heavy metal is 5–50 mg / L.

[0016] The concentration of the doxycycline is 20–200 mg / L.

[0017] This invention provides a strain of *Citrobacter muscaria* DH3, which has been biopreserved. The *Citrobacter muscaria* DH3 is tolerant to substances including Cu. 2+ Pb 2+ Cr 6+ Cd 2+It can remove multiple heavy metals, including doxycycline, and has a high efficiency in degrading doxycycline. Experiments have shown that the *Citrobacter murineis* strain DH3 described in this invention can tolerate dual pollution of heavy metals and doxycycline, and can simultaneously remove heavy metals and degrade doxycycline, providing a new strain resource for the treatment of aquaculture water pollution.

[0018] Biological Preservation Information

[0019] Citrobacter murliniae strain DH3 was deposited on February 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 29865. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 This is a colony diagram of strain DH3 from Example 2;

[0022] Figure 2 This is a SEM image of strain DH3 from Example 2;

[0023] Figure 3 This is the phylogenetic tree of strain DH3 in Example 2;

[0024] Figure 4 This illustrates the removal of different concentrations of doxycycline by strain DH3 in Example 3.

[0025] Figure 5 The strain DH3 in Example 4 is effective against the heavy metal Pb. 2+ and Cd 2+ Adsorption status;

[0026] Figure 6 The strain DH3 in Example 5 is effective against heavy metals (Pb). 2+ or Cd 2+ Removal characteristics of heavy metals and doxycycline in a complex pollution system containing doxycycline and doxycycline. Detailed Implementation

[0027] This invention provides a strain of Citrobacter murliniae, DH3, with accession number CGMCC No. 29865.

[0028] The strain DH3 described in this invention was isolated and screened from manure compost in a pig farm in Qibin District, Hebi City, Henan Province. It possesses tolerance and adsorption properties for substances including Cu. 2+ Pb 2+ Cr 6+ Cd 2+ This strain can detect and efficiently degrade multiple heavy metals, including doxycycline. Morphological and molecular biological identification confirmed it to be *Citrobacter murineis*, and it was named *Citrobacter murineis* strain DH3 and placed in biopreservation.

[0029] This invention also provides a microbial agent comprising *Citrobacter murineis* DH3 as described in the above-mentioned technical solution. As one embodiment, the OD of *Citrobacter murineis* DH3 in the microbial agent... 600 The concentration is 0.5–2; as another embodiment, the OD of Citrobacter murineis DH3 in the bacterial agent is... 600 The value is 1 to 1.5. In one embodiment, the microbial agent comprises the cells of *Citrobacter murineis* DH3 or the fermentation broth of *Citrobacter murineis* DH3.

[0030] In one embodiment, the preparation method of the bacterial agent of the present invention includes: inoculating *Citrobacter murineis* strain DH3 into a culture medium and culturing for 18-24 hours to obtain the bacterial agent. In one embodiment, the culture medium can be LB medium. In one embodiment, the temperature of the culturing can be 20-35°C; in another embodiment, the temperature of the culturing can be 30°C. In one embodiment, the rotation speed of the culturing can be 120-200 rpm; in another embodiment, the rotation speed of the culturing can be 150 rpm. In one embodiment, the culturing time can be 20-22 hours.

[0031] This invention also provides the application of the *Citrobacter murineis* strain DH3 or the bacterial agent described in the above-mentioned technical solutions in one or more of the following: treating aquaculture wastewater, removing heavy metals, and degrading doxycycline. As one embodiment, the treatment of aquaculture wastewater according to this invention includes removing heavy metals and / or doxycycline from the wastewater; as another embodiment, the treatment of aquaculture wastewater includes removing both heavy metals and doxycycline from the wastewater. As one embodiment, the heavy metal may be Pb. 2+ and / or Cd 2+ As another implementation, the heavy metal may be Pb. 2+ and Cd 2+In one embodiment, the concentration of the heavy metal can be 5–50 mg / L; in another embodiment, the concentration of the heavy metal can be 10–30 mg / L. In one embodiment, the concentration of doxycycline can be 20–200 mg / L; in another embodiment, the concentration of doxycycline can be 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, or 200 mg / L.

[0032] This invention also provides a method for reducing heavy metal and / or doxycycline pollution in aquaculture wastewater, comprising co-culturing *Citrobacter murineis* strain DH3 with aquaculture wastewater; the preservation number of *Citrobacter murineis* DH3 is CGMCC No. 29865; the aquaculture wastewater contains heavy metals and / or doxycycline. As one embodiment, based on the bacterial solution containing *Citrobacter murineis* DH3, the OD of the bacterial solution of *Citrobacter murineis* DH3 of this invention... 600 The OD value can be 0.5–2; as another embodiment, the OD value of the bacterial culture of *Citrobacter murineis* DH3 is... 600 The ratio can be 1 to 1.5. In one embodiment, the volume ratio of the *Citrobacter murineis* DH3 bacterial solution to the aquaculture wastewater can be 0.5 to 2:100. In another embodiment, the volume ratio of the *Citrobacter murineis* DH3 bacterial solution to the aquaculture wastewater can be 1:100. In one embodiment, the heavy metal can be Pb. 2+ and / or Cd 2+ As another implementation, the heavy metal may be Pb. 2+ and Cd 2+ In one embodiment, the concentration of the heavy metal can be 5–50 mg / L; in another embodiment, the concentration of the heavy metal can be 10–30 mg / L. In one embodiment, the concentration of doxycycline can be 20–200 mg / L; in another embodiment, the concentration of doxycycline can be 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, or 200 mg / L.

[0033] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] The culture media used in the following examples are as follows:

[0035] LB medium: NaCl 5g / L, yeast extract 10g / L, trypsin 10g / L, solvent is water, pH 7.0~.2.

[0036] Mineral culture medium: 1.5 g / L K2HPO4, 1.0 g / L NaCl, 0.5 g / L KH2PO4, 0.2 g / L MgSO4·7H2O (pH 7.0) and 10 g / L tryptone.

[0037] Solid mineral culture medium: The above mineral culture medium, agar 15g / L, solvent is water, pH 7.0-7.2.

[0038] Before using the above culture media in the following experiments, they were all placed in an autoclave and sterilized at 121°C for 30 minutes.

[0039] Example 1

[0040] Isolation, purification and screening of strains

[0041] (1) Isolation and purification of strains: Collect manure from a pig farm in Qibin District, Hebi City, Henan Province. Under aseptic conditions, add 3g of manure to 100mL of LB medium and place it in a constant temperature shaker at 30℃ and 150rpm for 48h in the dark to obtain the culture medium.

[0042] (2) Take 1 mL of the mixed culture medium from step (1) and add it to 100 mL of mineral culture medium containing 50 mg / L doxycycline. Place it in a constant temperature shaker at 30°C and 150 rpm and culture in the dark for 48 h to obtain a culture medium that is resistant to doxycycline.

[0043] (3) The culture medium obtained in step (2) that is resistant to doxycycline is prepared by mixing 10... -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Gradual dilution, taking 10... -4 10 -5 and 10 -6 100 μL of each of the three dilution gradients of culture medium was spread onto solid mineral culture medium and incubated upside down in an incubator at 30°C for 24–48 h.

[0044] (4) Number the colonies that grow in the solid mineral culture medium, and pick out the colonies separately for further streak isolation culture until purified single colonies are obtained;

[0045] (5) Strains were screened: Each single colony obtained in step (4) was inoculated into a mineral medium containing 50 mg / L doxycycline and its tolerance was examined.

[0046] (6) Select 5 strains with strong tolerance, pick strains from a single colony, inoculate them into LB medium for expansion culture, and place them in a constant temperature shaker at 30℃ and 150rpm for 20-24h in the dark.

[0047] (7) Adjust the bacterial concentration from the LB medium in step (6) to OD. 600 =1. Take 1 mL of the prepared bacterial suspension and add it to a mineral medium containing 50 mg / L doxycycline. Incubate in a constant temperature shaker at 30℃ and 150 rpm for 7 days in the dark. Use a mineral medium without bacterial suspension as a blank control. Set up 3 parallel experiments for each group. Take samples at fixed times every day and measure the biodegradation rate of doxycycline on days 0, 1, 2, 3, 4, 5, 6, and 7 to further investigate its biodegradation effect on doxycycline.

[0048] (8) Doxycycline sample preparation: Centrifuge the sample taken in step (7) at 8000 rpm for 10 min, draw up the supernatant with a syringe, filter it into a brown liquid phase sample bottle using a Φ0.22 μm organic filter membrane, and store it in a refrigerator at -20℃ for testing if it cannot be detected in time.

[0049] (9) Sample Detection: The concentration of doxycycline in the sample was determined using an Agilent 1260 high-performance liquid chromatograph. The sample injection volume was set to 20 μL; the mobile phase was 0.1% trifluoroacetic acid aqueous solution-acetonitrile-methanol, with a volume ratio of 61:26:13, and the flow rate was set to 1 mL / min; a ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm, Agilent) was used; the column oven temperature was set to 40℃; and the UV detector wavelength was 355 nm. The degradation rate of doxycycline was calculated according to Formula I.

[0050] Degradation rate of doxycycline (%) = (initial concentration - residual concentration) / initial concentration × 100% Formula I.

[0051] (10) The water samples of the five strains that were preserved were tested for degradation of doxycycline. Finally, it was determined that three strains could degrade 50 mg / L of doxycycline. On the 5th to 7th day, their total degradation rate of doxycycline was greater than 80%.

[0052] (11) The experimental conditions for investigating doxycycline / heavy metal co-contamination were: 50 mL mineral culture medium, heavy metal Cu 2+ Pb 2+ Cr 6+ Cd 2+ The concentrations were all 10 mg / L, and the concentration of doxycycline was 50 mg / L. OD was inoculated at a volume ratio of 1%. 600=1 In step (10), the bacterial cultures of the three strains were prepared, with mineral culture medium without inoculation of the strains as a blank control, and three parallel experiments were set up for each. They were placed in a constant temperature shaker at 30℃ and 150rpm in the dark for incubation. Sampling time was from 0 to 5 days, with sampling at 24-hour intervals.

[0053] (12) Analytical methods for four heavy metals: The sample was filtered through a 0.22 μm filter membrane and diluted by a certain factor. The Cu content in the sample was determined by flame atomic absorption spectrometry (Analytik Jena). 2+ Pb 2+ Cr 6+ Cd 2+ The content of.

[0054] (13) Finally, one strain was identified that could adsorb 10 mg / L of heavy metal Pb. 2+ Cd 2+ It also degrades 50 mg / L doxycycline, and this strain was named DH3.

[0055] Example 2

[0056] Identification of the DH3 strain obtained in Example 1

[0057] 1) Morphological identification of the DH3 strain obtained in Example 1: The DH3 strain was inoculated into solid agar medium and cultured at 30°C. The results showed that the DH3 strain grew into small, round, moist, and raised white colonies on the solid agar medium. Figure 1 As shown.

[0058] The micromorphology of strain DH3 was observed using SEM technology, and the results are shown in [Figure number missing]. Figure 2 The SEM image shows that DH3 is a bacillus, about 1-2 μm in length, with a relatively rough surface. This structure suggests that the surface of the strain may have a certain adsorption function.

[0059] Figure 3 As shown. Combined with Figure 1 and Figure 2 Based on the colony characteristics and phylogenetic tree results, the strain was identified as belonging to Citrobacter murliniae and named Citrobacter murliniae DH3. It was deposited at the China General Microbiological Culture Collection Center on February 4, 2024, with the accession number CGMCC No. 29865.

[0060] Example 3

[0061] The degradation effect of Citrobacter mellitus DH3 on different concentrations of doxycycline was investigated using the following steps:

[0062] (1) Activate the strain: Take the DH3 strain that has been frozen in glycerol and inoculate it into LB medium. After culturing in a shaker at 30℃ and 150rpm for 18-24h, take it out for use.

[0063] (2) Prepare a mineral culture medium with a peptone concentration of 8 g / L (the concentrations of other components remain unchanged) and a pH of 7. Dispense the culture medium into Erlenmeyer flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

[0064] (3) Add filtered sterilized doxycycline stock solution to the sterilized culture medium to make the initial doxycycline concentrations 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L and 200 mg / L respectively.

[0065] (4) Inoculate OD at a volume ratio of 1%. 600 =1 DH3 bacterial suspension was poured into an Erlenmeyer flask, and the culture medium without DH3 bacterial suspension was used as a hydrolysis control. Three parallel groups were set up for both the experimental group and the hydrolysis control group.

[0066] (5) Place the conical flask in a shaker at 25°C and incubate at 150 rpm. Take samples every 24 hours from 0 to 7 days.

[0067] (6) Centrifuge the extracted sample at 8000 rpm for 10 min, aspirate the supernatant with a syringe, filter it through a 0.22 μm organic filter membrane into a brown liquid phase sample bottle, and store it at -20℃ for later testing. The determination method is the same as step (9) in Example 1. Through previous dynamic observation, it was found that the hydrolysis and biodegradation of doxycycline reached equilibrium on day 5. The determination results on day 5, in which the biodegradation rate was calculated according to formula II.

[0068] Biodegradation rate = Total degradation rate of doxycycline - Hydrolysis rate (Formula II)

[0069] Wherein, hydrolysis rate = (initial doxycycline concentration - residual doxycycline concentration in the hydrolysis group) / initial doxycycline concentration × 100% Formula III;

[0070] Total degradation rate of doxycycline = (initial doxycycline concentration - residual doxycycline concentration in experimental group DH3) / initial doxycycline concentration × 100% Formula IV.

[0071] The results are shown in Table 1 and... Figure 4 .

[0072] Table 1. Results of hydrolysis and biodegradation rates of doxycycline at different concentrations.

[0073] Doxycycline concentration (mg / L) Hydrolysis rate (%) Biodegradation rate (%) 20 28.61 45.58 50 22.10 63.27 100 17.09 73.51 150 16.21 73.85 200 17.46 65.09

[0074] Depend on Figure 4 It can be concluded that the DH3 strain can grow and degrade doxycycline at concentrations of 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L and 200 mg / L. However, the growth and degradation of the DH3 strain are inhibited to varying degrees at high concentrations of doxycycline of 100 mg / L, 150 mg / L and 200 mg / L. The inhibitory effect is more obvious with increasing concentration. The biodegradation rate of doxycycline on day 5 ranges from 45.58% to 73.85%.

[0075] Example 4

[0076] The adsorption effect of DH3 strain on heavy metals was investigated using the following steps:

[0077] (1) Activate the strain: Take the DH3 strain that has been frozen in glycerol and inoculate it into LB medium. After culturing in a shaker at 30℃ and 150rpm for 18-24h, take it out for use.

[0078] (2) Prepare a mineral culture medium with a peptone concentration of 8 g / L and a pH of 7. Dispense the culture medium into Erlenmeyer flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

[0079] (3) Add the filtered and sterilized heavy metal mother liquor to the sterilized culture medium to reduce the initial heavy metal (Pb) content. 2+ or Cd 2+ The concentration is 10 mg / L.

[0080] (4) Inoculate OD at a volume ratio of 1%. 600 Pour DH3 bacterial suspension (=1) into an Erlenmeyer flask. Use culture medium without DH3 bacterial suspension as a blank control. Set up 3 parallel groups for both the experimental group and the blank control group.

[0081] (5) Place the conical flask in a shaker at 25°C and incubate at 150 rpm. Take samples every 24 hours for 0 to 3 days.

[0082] (6) After filtering the sample through a 0.22 μm filter membrane and diluting it by a certain factor, the Pb content in the sample was determined by flame atomic absorption spectrometry (Analytik Jena). 2+ and Cd 2+ The content of.

[0083] See results Figure 5 It can be seen that when the initial Pb 2+ Cd 2+ At a concentration of 10 mg / L, strain DH3 showed resistance to Pb after 72 hours. 2+ Cd 2+ The adsorption rates were 17.80% and 99.04%, respectively. In contrast, the adsorption rate of heavy metals in the blank control group was 0%.

[0084] Example 5

[0085] DH3 strain is resistant to doxycycline and heavy metals (Pb). 2+ Cd 2+ The degradation effect of compound pollution is assessed through the following steps:

[0086] (1) Activate the strain: Take the DH3 strain that has been frozen in glycerol and inoculate it into LB medium. After culturing in a shaker at 30℃ and 150rpm for 18-24h, take it out for use.

[0087] (2) Prepare a mineral culture medium with a peptone concentration of 8 g / L and a pH of 7. Dispense the culture medium into Erlenmeyer flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

[0088] (3) Add the filtered and sterilized heavy metals and doxycycline stock solution to the sterilized culture medium to reduce the initial heavy metal (Pb) content. 2+ or Cd 2+ The concentration was 10 mg / L, and the initial concentration of doxycycline was 50 mg / L.

[0089] (4) Inoculate OD at a volume ratio of 1%. 600 Pour DH3 bacterial suspension (=1) into an Erlenmeyer flask. Use culture medium without DH3 bacterial suspension as a blank control. Set up 3 parallel groups for both the experimental group and the blank control group.

[0090] (5) Place the conical flask in a shaker at 25°C and incubate at 150 rpm. Take samples every 24 hours for 0 to 3 days.

[0091] (6) Centrifuge the sample taken out at 8000 rpm for 10 min, aspirate the supernatant with a syringe, filter it into a brown liquid phase sample bottle using a Φ0.22 μm organic filter membrane, store it in a refrigerator at -20℃ for testing, and determine the degradation rate of doxycycline by DH3 strain after 72 h of treatment using the determination method in step (9) of Example 1.

[0092] The sample was filtered through a 0.22 μm filter membrane and diluted by a certain factor. The Pb content in the sample was then determined by flame atomic absorption spectrometry (AnalytikJena). 2+ and Cd 2+ The content of.

[0093] The results are shown in Table 2 and Figure 6 .

[0094] Table 2. Degradation effect of DH3 strain on combined doxycycline and heavy metal pollution (3 days)

[0095]

[0096]

[0097] The measurement results are as follows Figure 6 As shown: It can be seen that at 10 mg / L b 2+ Under combined pollution with 50 mg / L doxycycline, and with 10 mg / L Pb 2+ Compared to a single contamination system, the DH3 strain showed better protection against Pb after 72 hours. 2+ The removal rate increased to 30.04%; meanwhile, the removal rate of 10 mg / L Pb was also reduced. 2+ The DH3 strain had no significant effect on the biodegradation of doxycycline; under its presence, the biodegradation rate of doxycycline was 60.05%. (At 10 mg / L Cd...) 2+ Under combined pollution with 50 mg / L doxycycline and 10 mg / L Cd 2+ Compared to a single contamination system, the DH3 strain showed better protection against Cd after 72 hours. 2+ The removal rate showed no significant difference, with an adsorption rate of 98.33%; meanwhile, the removal rate of 10 mg / L Cd was also significantly reduced. 2+ It also had no significant effect on the biodegradation of doxycycline; under its presence, the biodegradation rate of doxycycline by the DH3 strain was 60.3%.

[0098] strain DH3 in heavy metal Pb 2+ Cd 2+ The degradation rates of 50 mg / L doxycycline under conditions of 5–50 mg / L are shown in Tables 3 and 4. Tables 3 and 4 show that the total degradation rate (the sum of hydrolysis and biodegradation rates) of doxycycline gradually decreases with increasing heavy metal concentration, and is related to the degradation rates of doxycycline and Cd.2+ Compared to combined contamination, strain DH3 is less susceptible to Pb contamination. 2+ It has strong adsorption properties and is effective against doxycycline and Pb. 2+ Under conditions of combined pollution, the degradation rate of doxycycline is better.

[0099] Table 3. Strain DH3 at Pb 2+ Cd 2+ Degradation rate of 50 mg / L doxycycline at (5–50 mg / L) concentrations (5 days)

[0100]

[0101] Table 4. Strain DH3 in Pb 2+ Cd 2+ Degradation rate of 50 mg / L doxycycline at (5-50 mg / L) concentrations (3 days)

[0102]

[0103] Comparative Example 1

[0104] DH3 strain is resistant to doxycycline and heavy metals (Cu). 2+ Cr 6+ The degradation effect of compound pollution is assessed through the following steps:

[0105] (1) Activate the strain: Take the DH3 strain that has been frozen in glycerol and inoculate it into LB medium. After culturing in a shaker at 30℃ and 150rpm for 18-24h, take it out for use.

[0106] (2) Prepare a mineral culture medium with a peptone concentration of 8 g / L and a pH of 7. Dispense the culture medium into Erlenmeyer flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

[0107] (3) Add the filtered and sterilized heavy metal mother liquor to the sterilized culture medium to reduce the initial heavy metal (Cu) content. 2+ Cr 6+ The concentration was 10 mg / L, and the initial concentration of doxycycline was 50 mg / L.

[0108] (4) Inoculate OD at a volume ratio of 1%. 600 =1 DH3 bacterial suspension was poured into Erlenmeyer flasks, with culture medium without DH3 bacterial suspension as a blank control. In this group, DH3 bacterial suspension was added to mineral culture medium containing only 50 mg / L as a control group. Each experimental group, blank control and control group were set up in 3 parallel groups.

[0109] (5) Place the conical flask in a shaker at 25°C and incubate at 150 rpm. Take samples every 24 hours for 0 to 3 days.

[0110] (6) Centrifuge the sample taken out at 8000 rpm for 10 min, draw the supernatant with a syringe, filter it into a brown liquid phase sample bottle using a Φ0.22 μm organic filter membrane, store it in a refrigerator at -20℃ for testing, and determine the degradation rate of doxycycline using the determination method in step (9) of Example 1.

[0111] The sample was filtered through a 0.22 μm filter membrane and diluted a certain factor. The concentration of Cu in the sample was then determined using a flame atomic absorption spectrometer (AnalytikJena). 2+ Cr 6+ The content of ).

[0112] The results show that when the initial (Cu) 2+ Cr 6+ When the concentration was 10 mg / L, the DH3 strain showed resistance to Cu after 3 days. 2+ Cr 6+ No adsorption occurred, and doxycycline reacted with Cu... 2+ and Cr 6+ Under coexisting conditions, the removal of doxycycline by strain DH3 was slightly inhibited, with a total degradation rate of 76.25% (Cu). 2+ ) and 72.79% (Cr 6+ The removal rate of Cu from the 50 mg / L doxycycline system by the DH3 strain in the control group was 78.83%, while that in the blank control group was much lower. 2+ When coexisting with doxycycline, the hydrolysis rate of doxycycline is 13.6%, Cr 6+ When coexisting with doxycycline, the hydrolysis rate of doxycycline was 14.06%. This indicates that the DH3 strain is resistant to Cu. 2+ and Cr 6+ It has a certain degree of tolerance, but it does not have adsorption properties.

[0113] From the above examples, it can be concluded that the *Citrobacter murineis* DH3 described in this invention also has the ability to adsorb Pb. 2+ and / or Cd 2+ For Cu 2+ Cr 6+ Pb 2+ and Cd 2+ It exhibits good tolerance and also possesses the biological function of degrading doxycycline, with excellent efficacy.

[0114] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of Citrobacter murine ( Citrobacter murliniae The strain DH3, with accession number CGMCCNo.29865, is used.

2. An inoculant characterized in that, The bacterial agent comprises the Citrobacter murliniae strain DH3 according to claim 1.

3. The bacterial agent of claim 2, wherein OD of the Enterococcus mundtii strain DH3 in the bacterial agent 600 is 0.5-2.

4. The bacterial agent of claim 2 or 3, characterized in that, The bacterial agent comprises the bacterial body of the Citrobacter murliniae strain DH3 or the fermentation liquor of the Citrobacter murliniae strain DH3.

5. Use of the strain of Citrobacter murltius DH3 according to claim 1 or of the inoculant according to any one of claims 2 to 4 for the removal of heavy metals from the environment or waste, the heavy metals being Pb 2+ and / or Cd 2+ .

6. Use according to claim 5, characterized in that, The waste comprises aquaculture wastewater.

7. Use according to claim 5 or 6, characterized in that, The concentration of the heavy metal is 5-50 mg / L. The concentration of the doxycycline is 20-200 mg / L.

8. A method of reducing contamination of heavy metals and / or doxycycline in aquaculture wastewater, characterized in that, The Citrobacter murltius strain DH3 is mixed with aquaculture wastewater for cultivation; the preservation number of the Citrobacter murltius strain DH3 is CGMCC No.29865, and the heavy metal is Pb 2+ and / or Cd 2+ .

9. The method of claim 8, wherein, The adding amount of the strain of Citrobacter murlae DH3 is calculated according to the volume ratio of the bacterial solution containing the strain of Citrobacter murlae DH3 to the aquaculture wastewater, which is (0.5-2):100; and the OD 600 of the bacterial solution of the strain of Citrobacter murlae DH3 is 0.5-2.

10. The method according to claim 8 or 9, characterized in that, The concentration of the heavy metal is 5-50 mg / L. The concentration of the doxycycline is 20-200 mg / L.

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