Citrobacter mussmusei strain DH3 and application thereof in adsorption of heavy metals and / or degradation of doxycycline

Abstract

The invention provides a Citrobacter muurlinae strain DH3 and an application of the Citrobacter muurlinae strain DH3 in adsorption of heavy metals and / or degradation of doxycycline, and belongs to the technical field of environmental microorganisms. The invention provides a Citrobacter mussmui strain DH3, and the Citrobacter mussmui strain DH3 has been biologically preserved. The citrobacter mussmui strain DH3 can tolerate various heavy metals including Cu < 2 + >, Pb < 2 + >, Cr < 6 + > and Cd < 2 + >, has efficient removal capacity on Pb < 2 + > and / or Cd < 2 + >, and has efficient degradation capacity on doxycycline at the same time. Tests show that the Citrobacter mussmusei strain DH3 can tolerate combined pollution conditions of heavy metals and doxycycline, can achieve the effects of removing the heavy metals and degrading the doxycycline at the same time, and provides a new strain resource for treatment of aquaculture water pollution.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microorganisms, and particularly relates to a Citrobacter muehii strain DH3 and its application in adsorbing heavy metals and / or degrading doxycycline. Background Art

[0002] Since heavy metals cannot be degraded by microorganisms and mostly exist stably in the environment in the form of precipitation, oxides, etc., and adhere to particles, it is difficult to fully contact and react the repair agent with the heavy metals during the repair process, resulting in difficult to control heavy metal pollution. Heavy metals are easily accumulated in organisms and enter the human body through the food chain, posing a serious threat to human health. Doxycycline wastewater is difficult to treat directly using biological methods because it contains high levels of suspended solids (SS) and residual antibiotics. When doxycycline remains in the environmental medium, it will be affected by biological or non-biological factors in the medium, causing its parent compound to change from a large molecule to a small molecule, eventually forming H2O and CO2. Due to the extremely complex influencing factors in the environmental medium, there are many different degradation methods, which are mainly divided into two categories: non-biological and biological degradation.

[0003] Existing methods for removing heavy metals and doxycycline each have their advantages, but they also suffer from disadvantages, 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. This refers to the metabolism of microorganisms in specific environments, which produces substances such as enzymes. This process directly or indirectly alters the parent structure of the antibiotic, thereby inactivating it. Consequently, an increasing number of microorganisms are being discovered for use in environmental pollutant remediation. Important strains, such as Bacillus and Pseudomonas, have been widely used to remove heavy metals from wastewater or soil due to their high metal-binding affinity. However, research on the microbial degradation of doxycycline is relatively limited. Furthermore, most studies focus on the removal of a single pollutant, with limited research on screening strains that demonstrate effective removal of both doxycycline and heavy metals. Summary of the Invention

[0004] The present invention aims to provide a Citrobacter rodentium strain DH3, which has the dual functions of adsorbing heavy metals and degrading doxycycline with excellent effects.

[0005] The present invention provides a Citrobacter murliniae strain DH3, with a deposit number of CGMCC No. 29865.

[0006] The present invention also provides a bacterial agent, which includes the Citrobacter muehii DH3 described in the above technical solution.

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

[0008] Preferably, the bacterial agent includes the bacterial cells of the Citrobacter muehii strain DH3 or the fermentation liquid of the Citrobacter muehii strain DH3.

[0009] The present invention also provides the use of the Citrobacter muehii strain DH3 described in the above technical solution or the bacterial agent described in the above technical solution in one or more of treating aquaculture wastewater, removing heavy metals and degrading doxycycline.

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

[0011] Preferably, the heavy metals include Pb 2+ and / or Cd 2+ ; The concentration of the heavy metal is 5 to 50 mg / L;

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

[0013] The present invention also provides a method for reducing heavy metal and / or doxycycline contamination in aquaculture wastewater, comprising mixing and culturing the Citrobacter muehii strain DH3 with the aquaculture wastewater; the deposit number of the Citrobacter muehii strain DH3 is CGMCC No. 29865.

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

[0015] Preferably, the heavy metals include Pb 2+ and / or Cd 2+ ; The concentration of the heavy metal is 5 to 50 mg / L;

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

[0017] The present invention provides a Citrobacter moorei strain DH3, and has completed biological preservation. The Citrobacter moorei DH3 can tolerate Cu 2+ , Pb 2+ Cr 6+ 、Cd 2+Experiments have shown that the Citrobacter rodentium strain DH3 described in the present invention can tolerate the dual pollution of heavy metals and doxycycline, and can simultaneously remove heavy metals and degrade doxycycline, providing a new bacterial strain resource for the treatment of aquaculture water pollution.

[0018] Biological deposit information

[0019] Citrobacter murliniae strain DH3 was deposited on February 4, 2024, at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCC No. 29865. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0021] Figure 1 This is the colony map of the strain DH3 in Example 2;

[0022] Figure 2 This is a SEM characterization image of the strain DH3 in Example 2;

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

[0024] Figure 4 The removal of different concentrations of doxycycline by strain DH3 in Example 3 is shown;

[0025] Figure 5 The strain DH3 in Example 4 is sensitive to heavy metal Pb 2+ and Cd 2+ Adsorption conditions;

[0026] Figure 6 The strain DH3 in Example 5 is sensitive to heavy metals (Pb 2+ or Cd 2+ ) and doxycycline complex pollution system. DETAILED DESCRIPTION

[0027] The present invention provides a Citrobacter murliniae strain DH3, with a deposit number of CGMCC No. 29865.

[0028] The strain DH3 of the present invention was isolated and screened from a pig farm in Qibin District, Hebi City, Henan Province, and has the ability to tolerate and adsorb Cu 2+ , Pb 2+ Cr 6+ 、Cd 2+ The strain was identified as Citrobacter muehii strain DH3 through morphological and molecular biological identification and was subsequently designated as Citrobacter muehii strain DH3.

[0029] The present invention also provides a bacterial agent, which includes the Citrobacter muehii DH3 described in the above technical solution. As an embodiment, the OD of Citrobacter muehii DH3 in the bacterial agent is 600 As another embodiment, the OD of Citrobacter moorei DH3 in the bacterial agent is 0.5 to 2. 600 As an embodiment, the bacterial agent includes the bacterial cells of the Citrobacter muehii DH3 or the fermentation liquid of the Citrobacter muehii DH3.

[0030] In one embodiment, the method for preparing the bacterial agent of the present invention comprises: inoculating Citrobacter rodentium strain DH3 into a culture medium and performing an expanded culture for 18 to 24 hours to obtain the bacterial agent. In one embodiment, the culture medium may be LB medium. In one embodiment, the temperature of the expanded culture may be 20 to 35°C; in another embodiment, the temperature of the expanded culture may be 30°C. In one embodiment, the rotation speed of the expanded culture may be 120 to 200 rpm; in another embodiment, the rotation speed of the expanded culture may be 150 rpm. In one embodiment, the expanded culture time may be 20 to 22 hours.

[0031] The present invention also provides the use of the Citrobacter muehri strain DH3 described in the above technical solution or the bacterial agent described in the above technical solution in one or more of treating aquaculture wastewater, removing heavy metals and degrading doxycycline. As an embodiment, the treatment of aquaculture wastewater in the present invention includes removing heavy metals and / or doxycycline in aquaculture wastewater; as another embodiment, the treatment of aquaculture wastewater includes removing heavy metals and doxycycline in aquaculture wastewater. As an embodiment, the heavy metal can be Pb 2+ and / or Cd 2+ As another embodiment, the heavy metal may be Pb 2+ and Cd 2+In one embodiment, the concentration of the heavy metal may be 5 to 50 mg / L; in another embodiment, the concentration of the heavy metal may be 10 to 30 mg / L. In one embodiment, the concentration of doxycycline may be 20 to 200 mg / L; in another embodiment, the concentration of doxycycline may be 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, or 200 mg / L.

[0032] The present invention also provides a method for reducing heavy metal and / or doxycycline contamination in aquaculture wastewater, wherein the Citrobacter muehii DH3 strain is mixed with aquaculture wastewater and cultured; the deposit number of the Citrobacter muehii DH3 is CGMCC No. 29865; and the aquaculture wastewater contains heavy metals and / or doxycycline. As an embodiment, based on the bacterial solution containing the Citrobacter muehii DH3, the OD value of the bacterial solution of the Citrobacter muehii DH3 of the present invention is 600 can be 0.5 to 2; as another embodiment, the OD of the bacterial solution of Citrobacter moorei DH3 600 As an embodiment, the volume ratio of the bacterial liquid of Citrobacter muehii DH3 to the aquaculture wastewater can be 0.5-2:100. As another embodiment, the volume ratio of the bacterial liquid of Citrobacter muehii DH3 to the aquaculture wastewater can be 1:100. As an embodiment, as an embodiment, the heavy metal can be Pb 2+ and / or Cd 2+ As another embodiment, the heavy metal may be Pb 2+ and Cd 2+ In one embodiment, the concentration of the heavy metal may be 5 to 50 mg / L; in another embodiment, the concentration of the heavy metal may be 10 to 30 mg / L. In one embodiment, the concentration of doxycycline may be 20 to 200 mg / L; in another embodiment, the concentration of doxycycline may be 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, or 200 mg / L.

[0033] 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.

[0034] The culture medium used in the following examples is 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 / LMgSO4·7H2O (pH 7.0) and 10 g / L tryptone.

[0037] Solid mineral culture medium: the above-mentioned mineral culture medium, 15 g / L agar, water as solvent, and a pH of 7.0-7.2.

[0038] Before being used in the following experiments, the above culture media were placed in a high-pressure steam sterilizer and sterilized at 121°C for 30 minutes.

[0039] Example 1

[0040] Isolation, purification and screening of strains

[0041] (1) Strain Isolation and Purification: Manure was collected from a pig farm in Qibin District, Hebi City, Henan Province. Under aseptic conditions, 3 g of manure was added to 100 mL of LB medium and incubated in a constant temperature shaker at 30°C and 150 rpm in the dark for 48 h to obtain a culture medium.

[0042] (2) taking 1 mL of the mixed culture medium in step (1) and adding it to 100 mL of a mineral culture medium containing 50 mg / L doxycycline, placing it in a constant temperature shaker at 30°C and 150 rpm in the dark and culturing it for 48 hours to obtain a culture medium with tolerance to doxycycline;

[0043] (3) The doxycycline-resistant culture solution obtained in step (2) was heated to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 Serial dilutions were made, 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 to 48 hours;

[0044] (4) Number the colonies grown on the solid mineral culture medium, pick out the colonies, and further isolate and culture them by streaking until a purified single colony is obtained;

[0045] (5) Strain screening: Each single colony obtained in step (4) was inoculated into a mineral culture medium containing 50 mg / L of doxycycline to examine its tolerance;

[0046] (6) Select five strains with strong tolerance, pick strains from single colonies, inoculate them into LB medium for expansion, and culture them in a constant temperature shaker at 30°C and 150 rpm in the dark for 20-24 h;

[0047] (7) Adjust the bacterial solution concentration in the LB medium from step (6) to OD 600 =1, take 1mL of the prepared bacterial solution and add it to a mineral culture medium containing 50mg / L of doxycycline. Incubate in a constant temperature shaker at 30°C and 150rpm in the dark for 7 days. Use a mineral culture medium without bacterial solution as a blank control. Set up three parallel experiments for each group. Samples were taken at a fixed time every day, and the biodegradation rate of doxycycline was measured on days 0, 1, 2, 3, 4, 5, 6, and 7 to further investigate the biodegradation effect of the bacterial solution on doxycycline.

[0048] (8) Doxycycline sample processing: The sample collected in step (7) was centrifuged at 8000 rpm for 10 min, the supernatant was aspirated with a syringe, and filtered through a Φ0.22 μm organic filter membrane into a brown liquid sample bottle. If it cannot be tested in time, it was stored in a refrigerator at -20°C for testing.

[0049] (9) Sample testing: The concentration of doxycycline in the samples was determined using an Agilent 1260 high-performance liquid chromatograph. The sample injection volume was set to 20 μL. The mobile phase consisted of 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. The chromatographic column used was a ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm, Agilent). The column oven temperature was set to 40°C, 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 degradation of doxycycline by the five preserved strains was determined in water samples. Finally, it was determined that three strains could degrade 50 mg / L of doxycycline, and their total degradation rate of doxycycline was greater than 80% on days 5 to 7;

[0052] (11) The conditions for the investigation of doxycycline / heavy metal combined pollution were: 50 mL mineral culture medium, heavy metal Cu 2+ , Pb 2+ Cr 6+ 、Cd 2+ The concentrations were 10 mg / L, the concentration of doxycycline was 50 mg / L, and the OD 600= 1, the bacterial suspensions of the three strains in step (10) were cultured in a mineral culture medium without the strains as a blank control. Three parallel experiments were performed in each group. The culture suspensions were placed in a constant temperature shaker at 30°C and 150 rpm in the dark. The samples were taken from 0 to 5 days at 24-hour intervals.

[0053] (12) The analysis method of four heavy metals was as follows: the sample was filtered through a 0.22 μm filter membrane and diluted to a certain multiple, and the Cu content in the sample was determined by flame atomic absorption spectrometry (Analytik Jena). 2+ , Pb 2+ Cr 6+ 、Cd 2+ content.

[0054] (13) Finally, a strain was identified that could adsorb 10 mg / L of heavy metal Pb 2+ 、Cd 2+ , and degraded 50 mg / L doxycycline at the same time. The 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 a solid agar medium and cultured at 30°C. The results showed that the DH3 strain grew as white colonies on the solid agar medium. The colonies were small, round, and had a moist, convex surface. Figure 1 shown.

[0058] The microscopic morphology of DH3 strain was observed by SEM technology. Figure 2 The SEM image shows that DH3 is a bacillus, about 1 to 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 Figure 1 and Figure 2 Based on the colony characteristics and phylogenetic tree results, it can be seen that the strain belongs to Citrobacter murliniae, and is named Citrobacter murliniae DH3. It was deposited in the China General Microbiological Culture Collection Center on February 4, 2024, with the deposit number CGMCC No. 29865.

[0060] Example 3

[0061] The degradation effect of Citrobacter rodentium DH3 on different concentrations of doxycycline was tested in the following steps:

[0062] (1) Activate the strain. Take the DH3 strain frozen in glycerol and inoculate it into LB medium. Incubate it in a shaker at 30°C and 150 rpm for 18-24 hours and then 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 conical flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

[0064] (3) Add filter-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 1% by volume 600 = 1 DH3 bacterial solution was transferred into a conical flask, and the culture medium without DH3 bacterial solution was used as the 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 culture at 150 rpm. The sampling time is 0 to 7 days, and the sampling is performed every 24 hours.

[0067] (6) The sample was centrifuged at 8000 rpm for 10 min, the supernatant was aspirated with a syringe, filtered through a Φ0.22 μm organic filter membrane into a brown liquid sample bottle, and stored in a refrigerator at -20°C for testing. The determination method was the same as step (9) in Example 1. Through preliminary dynamic observation, it was found that the hydrolysis and biodegradation of doxycycline reached equilibrium on the 5th day. The determination results on the 5th day, where 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 Hydrolysis rate and biodegradation rate 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 under doxycycline concentrations of 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L and 200 mg / L, but the growth and degradation of the DH3 strain are inhibited to varying degrees under high doxycycline concentrations of 100 mg / L, 150 mg / L and 200 mg / L. The inhibition becomes more obvious with increasing concentration. On the 5th day, the biodegradation rate of doxycycline is between 45.58% and 73.85%.

[0075] Example 4

[0076] The adsorption effect of DH3 strain on heavy metals is as follows:

[0077] (1) Activate the strain. Take the DH3 strain frozen in glycerol and inoculate it into LB medium. Incubate it in a shaker at 30°C and 150 rpm for 18-24 hours and then 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 conical flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

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

[0080] (4) Inoculate OD at 1% by volume 600 = 1 DH3 bacterial solution was transferred into a conical flask, and the culture medium without DH3 bacterial solution was used as a blank control. Three parallel groups were set up for both the experimental group and the blank control group.

[0081] (5) Place the conical flask in a shaker at 25°C and culture at 150 rpm. The sampling time is 0 to 3 days, and the sampling is performed once every 24 hours.

[0082] (6) The sample was filtered through a 0.22 μm filter membrane and diluted to a certain multiple, and the Pb content in the sample was determined by flame atomic absorption spectrometry (Analytik Jena). 2+ and Cd 2+ content.

[0083] See the results Figure 5 . It can be seen that when the initial Pb 2+ 、Cd 2+ When the concentration of Pb was 10 mg / L, the strain DH3 had a strong effect on Pb 2+ 、Cd 2+ The adsorption rates of heavy metals in the blank control group were 17.80% and 99.04% respectively, while the heavy metal adsorption rate in the blank control group was 0.

[0084] Example 5

[0085] DH3 strains were sensitive to doxycycline and heavy metals (Pb 2+ 、Cd 2+ ) The degradation effect of composite pollution, the steps are as follows:

[0086] (1) Activate the strain. Take the DH3 strain frozen in glycerol and inoculate it into LB medium. Incubate it in a shaker at 30°C and 150 rpm for 18-24 hours and then 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 conical flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

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

[0089] (4) Inoculate OD at 1% by volume 600 = 1 DH3 bacterial solution was transferred into a conical flask, and the culture medium without DH3 bacterial solution was used as a blank control. Three parallel groups were set up for both the experimental group and the blank control group.

[0090] (5) Place the conical flask in a shaker at 25°C and culture at 150 rpm. The sampling time is 0 to 3 days, and the sampling is performed once every 24 hours.

[0091] (6) The sample was centrifuged at 8000 rpm for 10 min, the supernatant was aspirated with a syringe, filtered through a Φ0.22 μm organic filter membrane into a brown liquid sample bottle, and stored in a refrigerator at -20°C for testing. The degradation rate of doxycycline by the DH3 strain after 72 h of treatment was determined using the determination method in step (9) of Example 1;

[0092] The sample was filtered through a 0.22 μm filter membrane and diluted to a certain multiple, and the Pb content in the sample was determined by flame atomic absorption spectrometry (Analytik Jena). 2+ and Cd 2+ content.

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

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

[0095]

[0096]

[0097] The results of the test are as follows Figure 6 Shown: Visible, at 10mg / LPb 2+ and 50mg / L doxycycline combined pollution, and 10mg / LPb 2+ Compared with the single pollution system, the DH3 strain had a higher sensitivity to Pb at 72h. 2+ The removal rate of Pb increased to 30.04%; at the same time, the removal rate of 10 mg / L Pb 2+ There was no significant effect on the biodegradation of doxycycline. Under the presence of Cd, the biodegradation rate of doxycycline by DH3 strain was 60.05%. 2+ and 50mg / L doxycycline combined pollution, and 10mg / L Cd 2+ Compared with the single pollution system, the DH3 strain had a higher sensitivity to Cd at 72h. 2+ There was no significant difference in the removal rate of Cd, and the adsorption rate was 98.33%. 2+ It also had no significant effect on the biodegradation of doxycycline. In the presence of doxycycline, the biodegradation rate of doxycycline by DH3 strain was 60.3%.

[0098] strain DH3 in heavy metal Pb 2+ 、Cd 2+ The results of the degradation rate of 50 mg / L doxycycline under the conditions of (5-50 mg / L) are shown in Tables 3 and 4. As shown in Tables 3 and 4, with the increase of heavy metal concentration, the total degradation rate of doxycycline (the sum of hydrolysis rate and biodegradation rate) gradually decreased, and the degradation rate of doxycycline and Cd decreased with the increase of heavy metal concentration.2+ Compared with the combined pollution, the strain DH3 was more sensitive to Pb 2+ The adsorption capacity is strong, and it is suitable for doxycycline and Pb 2+ Under the condition of complex pollution, the degradation rate of doxycycline is better.

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

[0102]

[0103] Comparative Example 1

[0104] DH3 strains were sensitive to doxycycline and heavy metals (Cu 2+ Cr 6+ ) The degradation effect of composite pollution, the steps are as follows:

[0105] (1) Activate the strain. Take the DH3 strain frozen in glycerol and inoculate it into LB medium. Incubate it in a shaker at 30°C and 150 rpm for 18-24 hours and then 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 conical flasks, 50 mL per flask, and sterilize at 121°C for 25 min.

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

[0108] (4) Inoculate OD at 1% by volume 600 = 1 DH3 bacterial solution was added to a conical flask, and the culture medium without DH3 bacterial solution was used as a blank control. This group was further supplemented with DH3 bacterial solution and added with mineral culture medium containing only 50 mg / L as a control group. Three parallel groups were set up for the experimental group, blank control group and control group.

[0109] (5) Place the conical flask in a shaker at 25°C and culture at 150 rpm. The sampling time is 0 to 3 days, and the sampling is performed once every 24 hours.

[0110] (6) The sample was centrifuged at 8000 rpm for 10 min, the supernatant was aspirated with a syringe, filtered through a Φ0.22 μm organic filter membrane into a brown liquid sample bottle, and stored in a refrigerator at -20°C for testing. The degradation rate of doxycycline was determined using the determination method in step (9) of Example 1;

[0111] The sample was filtered through a 0.22 μm filter membrane and diluted to a certain multiple. The content of Cu in the sample was determined by flame atomic absorption spectrometry (Analytik Jena). 2+ Cr 6+ ) content.

[0112] The results show that when the initial (Cu 2+ Cr 6+ ) was 10 mg / L, the DH3 strain was sensitive to Cu 2+ Cr 6+ No adsorption, while doxycycline was adsorbed with Cu 2+ and Cr 6+ Under the coexistence conditions, the removal of doxycycline by DH3 strain was slightly inhibited, and the total degradation rate of doxycycline was 76.25% (Cu 2+ ) and 72.79% (Cr 6+ The removal rate of DH3 strain in the control group for 50 mg / L doxycycline system was 78.83%, while the removal rate of Cu in the blank control group was 1.3%. 2+ When coexisting with doxycycline, the hydrolysis rate of doxycycline was 13.6%, and Cr 6+ When coexisting with doxycycline, the hydrolysis rate of doxycycline was 14.06%. This shows that the DH3 strain is sensitive to Cu 2+ and Cr 6+ It has a certain tolerance but no adsorption.

[0113] From the above examples, it can be concluded that the Citrobacter moorei DH3 of the present invention has the ability to adsorb Pb 2+ and / or Cd 2+ , for Cu 2+ Cr 6+ , Pb 2+ and Cd 2+ It has good tolerance and the biological function of degrading doxycycline with excellent effect.

[0114] 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 Citrobacter murliniae strain DH3, deposited with CGMCC No. 29865.

2. A bacterial agent, characterized in that The bacterial agent includes the Citrobacter muehii strain DH3 according to claim 1.

3. The microbial agent according to claim 2, characterized in that The OD of the Citrobacter moorei strain DH3 in the bacterial agent 600 It is 0.5 to 2.

4. The bacterial agent according to claim 2 or 3, characterized in that The bacterial agent includes the bacterial body of the Citrobacter muehii strain DH3 or the fermentation liquid of the Citrobacter muehii strain DH3.

5. Use of the Citrobacter muehii strain DH3 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 for removing heavy metals from the environment or waste and / or degrading doxycycline in the environment or waste.

6. The use according to claim 5, characterized in that The waste includes aquaculture wastewater.

7. The use according to claim 5 or 6, characterized in that The heavy metals include Pb 2+ and / or Cd 2+ ; The concentration of the heavy metal is 5 to 50 mg / L; The concentration of the doxycycline is 20-200 mg / L.

8. A method for reducing heavy metal and / or doxycycline pollution in aquaculture wastewater, characterized in that: The Citrobacter muehii strain DH3 is mixed with aquaculture wastewater and cultured; the preservation number of the Citrobacter muehii strain DH3 is CGMCC No. 29865.

9. The method according to claim 8, characterized in that The addition amount of the Citrobacter muehii strain DH3 is calculated based on the bacterial solution containing the Citrobacter muehii strain DH3, and the volume ratio of the bacterial solution of the Citrobacter muehii DH3 to the aquaculture wastewater is (0.5-2):100; the OD of the bacterial solution of the Citrobacter muehii DH3 is 600 It is 0.5 to 2.

10. The method according to claim 8 or 9, characterized in that The heavy metals include Pb 2+ and / or Cd 2+ ; The concentration of the heavy metal is 5 to 50 mg / L; The concentration of the doxycycline is 20-200 mg / L.

Images