Providencia alkali-producing, bacterial agent, and application thereof

Abstract

The invention discloses a Providencia alcalifaciens, a bacterial agent, and an application thereof, which belongs to the field of bioengineering technology, wherein Providencia alcalifaciens (Providencia alcalifaciens SK-R-073 (H7)) is named H7, classified as Providencia alcalifaciens SK-R-073 (H7), and is deposited in the China Center for Type Culture Collection on October 20, 2022, with a preservation address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, zip code 430072, and a preservation number of CCTCC NO: M 20221613. The Providencia alcalifaciens provided by the invention can degrade heavy metals, control heavy metal pollution in the environment, and does not produce secondary pollution.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to Providencia alkali-producing, a bacterial agent, and applications thereof. Background Art

[0002] Providencia alcalifaciens belongs to the genus Providencia of the family Enterobacteriaceae. It is a Gram-negative, facultatively anaerobic bacterium that does not cluster, moves with peritrichous flagella, is oxidase-negative, and is catalase-positive. It is widely distributed in water bodies and soil in nature, and is also commonly found in aquatic animals, mammals, and human intestines. It usually infects humans through water, fish, and shellfish, and can produce virulence factors such as heat-stable and heat-labile enterotoxins, cytotoxins, hemolysins, and hemagglutinins. Studies have shown that the bacterium is a conditional pathogen that mainly causes infectious diarrhea, food poisoning, and sepsis in humans.

[0003] Environmental heavy metal pollution is becoming increasingly serious with the development of industrialization. Heavy metals are not only harmful to ecosystems and agricultural production, but also accumulate in animals and plants through the food chain, thus endangering human health. Heavy metals are difficult to remove from the environment. The toxicity of heavy metals is closely related to their chemical form. Therefore, changing the chemical form of heavy metals to reduce their toxicity is an important means of heavy metal pollution control. Microorganisms in the environment play an important role in the transformation of various heavy metal forms. Summary of the invention

[0004] The invention provides Providencia alkaligenes, a bacterial agent, and applications thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] In the first aspect, a Providencia alcalifaciens SK-R-073 (H7) is provided, which is named H7 and classified as Providencia alcalifaciens SK-R-073 (H7). It was preserved in the China Center for Type Culture Collection on October 20, 2022, and the preservation address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, postal code 430072, and the preservation number is CCTCC NO: M 20221613.

[0007] In a second aspect, a bacterial agent comprising the above-mentioned Providencia alcaligenes is provided.

[0008] In a third aspect, a use of Providencia alcaligenes or a bacterial agent in detoxifying heavy metals in the environment (soil and / or sewage) is provided.

[0009] Further, the heavy metal includes at least one of Hg, Se, Sb and As.

[0010] The beneficial effects of the present invention are:

[0011] The Providencia alcalifaciens SK-R-073 (H7) provided by the present invention can tolerate high concentrations of heavy metals, especially Se, Sb, Hg, and As, and can survive in an environment seriously polluted by heavy metals, such as soil or sewage, remove heavy metals from the environment, such as soil or sewage, and repair the environment without generating secondary pollution. It is of great significance to promoting human health, environmental protection, and sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A schematic diagram of the growth curve of strain H7 provided in Example 2 under stress of different concentrations of Se;

[0013] Figure 2 A schematic diagram of the growth curve of strain H7 provided in Example 2 under stress of different concentrations of Sb;

[0014] Figure 3 A schematic diagram of the growth curve of strain H7 provided in Example 2 under stress of different Hg concentrations;

[0015] Figure 4 A schematic diagram of the growth curve of strain H7 provided in Example 2 under different concentrations of As stress;

[0016] Figure 5 This is a schematic diagram of the Hg volatilization curve of strain H7 provided in Example 2 at different time points. DETAILED DESCRIPTION

[0017] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0018] Example 1

[0019] 1. Isolation and culture conditions

[0020] Culture medium: LB broth and LB agar

[0021] Medium ingredients: tryptone (10 g / L), yeast extract powder (5 g / L), sodium chloride (10 g / L), pH = 7.0 ± 0.1 (25 ° C)

[0022] Isolation medium: LB agar medium containing mercury (mercury concentration = 100 mg / L)

[0023] Culture conditions: anaerobic, 25°C

[0024] 2. Isolation of Hg-tolerant anaerobic microorganisms in paddy soil

[0025] 1. Prepare anaerobic liquid culture medium: Weigh an appropriate amount of LB culture medium, dissolve it with oxygen-free water, and then add 0.1% resazurin solution. Take a clean 30mL serum bottle, fill 10mL of LB culture medium into the bottle under nitrogen conditions, then seal it with a rubber stopper and an aluminum cap, and then sterilize it at 121℃ for 30min for later use.

[0026] 2. Prepare heavy metal tolerant culture medium: Weigh an appropriate amount of mercuric chloride powder (HgCl2), dissolve it in oxygen-free water, and add it to the culture medium in step 1 so that the Hg concentration in the culture medium is 100 mg / L, that is, Hg-containing culture medium.

[0027] 3. Deoxygenation: Sterilize the items needed for the experiment and place them in an anaerobic glove box.

[0028] 4. Dilution: Add 1 g of paddy soil collected from the four rice fields in Wanshan mercury mining area, Guizhou Province, to 9 mL (add a few glass beads during vortexing) of LB liquid culture medium, mix by vortexing for 15 minutes, and then place it in an anaerobic glove box for 20 minutes to allow the soil to settle and obtain the supernatant.

[0029] 5. Inoculation: Use a sterile syringe to take 100 μL of the supernatant in step 4 and add it to the Hg-containing culture medium in step 2, 2 parallels per group + a control group (without supernatant) (3×3=9 bottles); after culturing at 25°C for 3 days, the culture medium becomes turbid (the control group is not turbid).

[0030] 6. Re-inoculation: Use a sterile syringe to take 100 μL of the turbid solution in step 5 and inoculate it into a new Hg-containing culture medium (to confirm the bacteria's tolerance to mercury) and culture it at 25°C.

[0031] 7. Dilution: dilute the second inoculated bacterial solution in step 6 by taking 100 μL of bacterial solution into 900 μL of sterile water and mixing (10 -1 ), and then diluted step by step until 10 -6 .

[0032] 8. Spreading: Use tweezers to hold small glass beads and place them on the LB agar plate. Take 200 μL of the diluent and add it to the plate, then gently shake the plate to allow the glass beads to evenly spread the diluent to every corner of the plate. After spreading, seal the plate with sealing film (to ensure that the water in the plate does not lose), and culture at 25°C. Make two parallels for each concentration (6×2=36).

[0033] 9. First streaking: After 2-3 days of culture, the microorganisms begin to grow into colonies that can be identified by the naked eye, and single colonies can be selected for streaking. Use a disposable inoculation loop (1μL) to streak the colonies (three-zone streaking method), try not to select duplicate colonies, and record the characteristics of the colonies.

[0034] 10. Second streaking: Wait until the colonies from the first streaking have grown well (2 days later), then do the second streaking.

[0035] 11. Enrichment: After confirming that the colony is pure (i.e., there are no foreign bacteria growing together), use a 1μL inoculation loop to pick up a point of pure colony and inoculate it into liquid LB for enrichment (in a 2mL centrifuge tube), then use a syringe to take the bacterial solution and inject it into a serum bottle and culture it at 25℃.

[0036] 12. Seed preservation: Add 0.3mL-0.5mL sterile glycerol to a 2mL cryovial, add 1mL bacterial solution and mix well. Store at room temperature for 30 minutes (to ensure that the glycerol enters the cells) and then store at -80℃.

[0037] 3. Identification of Providencia alcaligenes

[0038] Identification: 16S rDNA sequence is shown in SEQ ID NO.1:

[0039]

[0040] After 16S rDNA sequencing analysis, the strain was identified as Providencia and presumably Providencia alcalifaciens H7.

[0041] Example 2

[0042] 1. Experiment on the tolerance of strain H7 to Se:

[0043] 1. Experimental design: Design 12 tolerance solution concentration gradients:

[0044] Se: 0, 100, 500, 1000, 2000, 4000, 6000, 8000, 10000, 80000, 120000mg / L.

[0045] 2. Preparation of tolerance solution: Weigh an appropriate amount of sodium selenite (Na2SeO3) powder, prepare it with LB liquid culture medium, and filter it with a 0.22μm filter membrane after it is fully dissolved and set aside.

[0046] 3. Cultivation: Take a sterile 96-well plate, the total system is 200 μL system, 180 μL culture medium + 20 μL bacterial solution, so that the heavy metal concentration of the system is as shown in step 1. After the inoculation is completed, add 50 μL sterile paraffin oil (sealed to isolate oxygen) to each well.

[0047] 4. Determination of growth curve: After the bacterial solution is inoculated, the 96-well plate is sealed with a sealing film (to prevent foreign matter from falling into the system), and the OD at time 0 is measured using an ELISA reader (model Thermo Scientific Multiskan FC). 600 The OD value was measured at regular intervals. 600 The growth curve of strain H1 under different concentrations of Se stress was plotted. Figure 1 As shown in the figure, Providencia alcaligenes can still grow under the condition of Se concentration of 0-80000 mg / L. As the concentration increases, the growth becomes slower, and the highest tolerance dose is Se concentration of 80000 mg / L.

[0048] 2. Experiment on the tolerance of strain H7 to Sb:

[0049] 1. Experimental design: Design 11 tolerance solution concentration gradients:

[0050] Sb: 0, 100, 400, 800, 1000, 2000, 4000, 6000, 8000, 10000, 18000mg / L.

[0051] 2. Preparation of tolerance solution: weigh an appropriate amount of potassium antimony tartrate (C8H4K2O12 Sb2·3H2O) powder was prepared with LB liquid culture medium, and after fully dissolved, it was filtered with a 0.22μm filter membrane for use.

[0052] 3. Cultivation: Take a sterile 96-well plate, the total system is 200 μL system, 180 μL culture medium + 20 μL bacterial solution, so that the heavy metal concentration of the system is as shown in step 1. After the inoculation is completed, add 50 μL sterile paraffin oil (sealed to isolate oxygen) to each well.

[0053] 4. Determination of growth curve: After the bacterial solution is inoculated, the 96-well plate is sealed with a sealing film (to prevent foreign matter from falling into the system), and the OD at time 0 is measured using an ELISA reader (model Thermo Scientific Multiskan FC). 600 The OD value was measured at regular intervals. 600 The growth curve of strain H1 under different concentrations of Sb stress was plotted. Figure 2 As shown in the figure, Providencia alcaligenes can still grow under the condition of Sb concentration of 0-18000g / L. As the concentration increases, the growth becomes slower, and the highest tolerance dose is Sb concentration of 17500g / L.

[0054] 3. Experiment on the tolerance of strain H7 to Hg:

[0055] 1. Experimental design: Design 12 tolerance solution concentration gradients:

[0056] Hg: 0, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300mg / L.

[0057] 2. Preparation of tolerance solution: Weigh an appropriate amount of mercuric chloride (HgCl2) powder, prepare it with LB liquid culture medium, and filter it with a 0.22μm filter membrane after it is fully dissolved and set aside.

[0058] 3. Cultivation: Take a sterile 96-well plate, the total system is 200 μL system, 180 μL culture medium + 20 μL bacterial solution, so that the heavy metal concentration of the system is as shown in step 1. After the inoculation is completed, add 50 μL sterile paraffin oil (sealed to isolate oxygen) to each well.

[0059] 4. Determination of growth curve: After the bacterial solution is inoculated, the 96-well plate is sealed with a sealing film (to prevent foreign matter from falling into the system), and the OD at time 0 is measured using an ELISA reader (model Thermo Scientific Multiskan FC). 600 The OD value was measured at regular intervals. 600 The growth curve of strain H1 under different Hg concentrations was plotted. Figure 3Providencia alcaligenes can still grow under the condition of Hg concentration of 0-300 mg / L. As the concentration increases, the growth becomes slower, and the highest tolerance dose is Hg concentration of 300 mg / L.

[0060] 4. Experiment on the tolerance of strain H7 to As:

[0061] 1. Experimental design: Design 12 tolerance solution concentration gradients:

[0062] As: 0, 100, 500, 1000, 2000, 40000, 6000, 8000, 10000, 30000, 50000, 80000mg / L.

[0063] 2. Preparation of tolerance solution: Weigh an appropriate amount of sodium arsenite (NaAsO2) powder, prepare it with LB liquid culture medium, and filter it with a 0.22μm filter membrane after it is fully dissolved and set aside.

[0064] 3. Cultivation: Take a sterile 96-well plate, the total system is 200 μL system, 180 μL culture medium + 20 μL bacterial solution, so that the heavy metal concentration of the system is as shown in step 1. After the inoculation is completed, add 50 μL sterile paraffin oil (sealed to isolate oxygen) to each well.

[0065] 4. Determination of growth curve: After the bacterial solution is inoculated, the 96-well plate is sealed with a sealing film (to prevent foreign matter from falling into the system), and the OD at time 0 is measured using an ELISA reader (model Thermo Scientific Multiskan FC). 600 The OD value was measured at regular intervals. 600 The growth curve of strain H1 under different concentrations of As stress was plotted. Figure 4 As shown in the figure, Providencia alcaligenes can still grow under the condition of Hg concentration of 0-30000 mg / L. As the concentration increases, the growth becomes slower, and the highest tolerance dose is As concentration of 30000 mg / L.

[0066] V. Experiment on Hg volatilization of strain H7:

[0067] 1. Experimental design: Determination of mercury volatilization at 5 time points

[0068] Time: 0h, 9h, 24h, 48h, 58h

[0069] 2. Preparation of mercury volatilization culture medium: Weigh an appropriate amount of mercuric chloride (HgCl2) powder, prepare it with LB liquid culture medium, and filter it with a 0.22μm filter membrane to sterilize it after it is fully dissolved.

[0070] 3. Cultivation: Take a sterile anaerobic borosilicate glass bottle, add 1.8mL culture medium and 200μL bacterial solution, the concentration of mercury is 10ng / mL, after inoculation, culture at 25℃ under anaerobic dark conditions.

[0071] 4. Determination of Hg volatilization rate: At the incubation time point in step 1, use sterile nitrogen to capture the mercury in the gas phase above the anaerobic bottle into a gold tube with a strong adsorption capacity for mercury, and then determine the concentration of captured mercury, which is the volatilization amount of mercury (such as Figure 5 ).

[0072] The Providencia alcalifaciens provided by the present invention can tolerate high concentrations of heavy metals, especially Se, Sb, Hg, and As, and can survive in an environment seriously polluted by heavy metals, such as soil or sewage, remove heavy metals from the environment, such as soil or sewage, and repair the environment without generating secondary pollution. It is of great significance to promoting human health, environmental protection, and sustainable agricultural development.

[0073] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0074] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A Providencia alcaligenes ( Providencia alcalifaciens )SK-R-073 (H7), characterized in that, The alkali-producing Providencia was deposited in the China Center for Type Culture Collection on October 20, 2022, with the deposit address being Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with a postal code of 430072, and the deposit number being CCTCC NO: M20221613.

2. A bacterial agent, characterized in that The bacterial agent includes the Providencia alcaligenes described in claim 1.

3. Use of the Providencia alcaligenes described in claim 1 or the bacterial agent described in claim 2 in repairing a Hg-contaminated environment.

4. The use according to claim 3, characterized in that: The environment includes soil and / or sewage.

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