Method for inactivating viruses in sewage water

Abstract

The application relates to a method for inactivating viruses in sewage. The method comprises the following steps: introducing heterotrophic bacteria into sewage to be inactivated; increasing the carbon-nitrogen ratio of the sewage during the activation and expansion of the heterotrophic bacteria; washing and resuspending the activated and expanded heterotrophic bacteria to form a heterotrophic bacteria liquid to be used; inoculating the heterotrophic bacteria liquid into the sewage and reducing the carbon-nitrogen ratio of the sewage, so that the heterotrophic bacteria produce an oxidative stress response to inactivate viruses. The method adjusts the carbon-nitrogen ratio to make the heterotrophic bacteria change from an eutrophic environment to an oligotrophic environment, activates the oxidative stress pathway of the heterotrophic bacteria, increases the generation of oxidative active species such as superoxide free radicals, causes oxidative damage to the virus shell or envelope, and thus promotes the inactivation of viruses. The method is simple and efficient, and has a wide application scenario.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sewage treatment, and in particular to a method for inactivating viruses in sewage. BACKGROUND

[0002] In recent years, the removal of pathogenic microorganisms such as viruses has become a major challenge to global public health and a hot issue in the field of sewage treatment. Traditional virus inactivation methods include heat inactivation, chemical inactivation and physical inactivation, etc. However, these methods have problems such as high cost, low inactivation rate and secondary environmental hazards.

[0003] Therefore, there is a need for an efficient and environmentally friendly method for inactivating viruses in sewage. SUMMARY

[0004] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0005] The present application provides a method for inactivating viruses in sewage, comprising introducing heterotrophic bacteria into the sewage to be inactivated; increasing the carbon-nitrogen ratio of the sewage during the activation and cultivation of the heterotrophic bacteria; washing and resuspending the activated and cultivated heterotrophic bacteria to form a heterotrophic bacteria solution for use; inoculating the heterotrophic bacteria solution into the sewage and reducing the carbon-nitrogen ratio of the sewage, so that the heterotrophic bacteria produce an oxidative stress response to inactivate the viruses.

[0006] In the present application, the term "sewage" can also be referred to as wastewater, which generally includes industrial wastewater, discharged domestic sewage, agricultural wastewater, etc., but does not contain strong toxic or strong oxidizing chemicals such as halogen acetic acid, hydrogen peroxide, or the content thereof does not affect the activity of microorganisms. In the present application, the initial carbon-nitrogen ratio of the sewage can be not more than 3.0, such as in the range of 0.5-3.0.

[0007] In the present application, the sewage can include carbon sources, nitrogen sources and trace elements. The carbon source can include but is not limited to at least one of sodium acetate, sodium lactate, sodium citrate, glucose and glycerol. The nitrogen source can include but is not limited to at least one of nitrate (such as sodium nitrate), nitrite, peptone, ammonium chloride. The trace elements include but are not limited to at least one of MgSO4, Na2-EDTA, FeCl3·6H2O, MnCl2·4H2O, Na2MoO4·2H2O, CuCl2·2H2O, CaCl 2、 ZnCl2.

[0008] In an embodiment, the activating and propagating the heterotrophic bacteria can comprise: performing a first activation and propagation of the heterotrophic bacteria in the wastewater having an initial carbon to nitrogen ratio of less than or equal to 3.0; and performing a second activation and propagation of the heterotrophic bacteria in the wastewater having an increased carbon to nitrogen ratio in the range of 5.0-10.0.

[0009] In an embodiment, the first activation and propagation is performed for a time period in the range of 6-30 h.

[0010] In an embodiment, the second activation and propagation is performed for a time period in the range of 6-30 h.

[0011] In an embodiment, both the first activation and propagation and the second activation and propagation are performed at a temperature in the range of 25-37 °C.

[0012] In an embodiment, the reducing the carbon to nitrogen ratio of the wastewater comprises reducing the carbon to nitrogen ratio of the wastewater to a range of 0.2-1.5:1.

[0013] In an embodiment, the heterotrophic bacteria is selected from one or more of Paracoccus denitrificans (P. denitrificans) Paracoccus denitrificans ), Bacillus subtilis (B. subtilis) Bacillus subtilis ), and Geobacter sulfurreducens (G. sulfurreducens) Geobacter sulfurreducens pca ).

[0014] In an embodiment, the virus is selected from one or more of Φ6 bacteriophage (Φ6) Phi 6 bacteriophage ), MS2 bacteriophage (MS2) MS2 bacteriophage ), T4 bacteriophage (T4) T4 bacteriophage ), and T7 bacteriophage (T7) T4 bacteriophage ).

[0015] In an embodiment, the wastewater comprises a carbon source, a nitrogen source, and trace elements.

[0016] In an embodiment, the carbon source is selected from one or more of sodium acetate, sodium citrate, glycerol, and glucose.

[0017] In an embodiment, the nitrogen source is selected from one or more of sodium nitrate, proteose peptone, and ammonium chloride.

[0018] In an embodiment, the trace elements are selected from one or more of MgS04, Na2-EDTA, FeCl3-6H20, MnCl2-4H20, Na2Mo04-2H20, CuCl2-2H20, CaCl2, and ZnCl2. Alternatively, the trace elements are selected from two or more of MgS04, Na2-EDTA, FeCl3-6H20, MnCl2-4H20, Na2Mo04-2H20, CuCl2-2H20, CaCl2, and ZnCl2.

[0019] In an embodiment, the buffer solution used to adjust the pH of the sewage can be a phosphate buffer solution.

[0020] In an embodiment, after the activation and expansion of the heterotrophic bacteria, the heterotrophic bacteria are washed and resuspended with a biological buffer solution; optionally, the biological buffer solution is selected from at least one of a Tris-HCl buffer solution, a HEPES buffer solution, and a PBS buffer solution; optionally, the concentration of the biological buffer solution is 20-100 mM; optionally, the pH of the biological buffer solution is 7.0-7.6.

[0021] In an embodiment, the inoculation concentration OD 600 In the range of 0.1-0.5.

[0022] In another aspect, the present application provides a method for inactivating viruses in sewage, comprising introducing heterotrophic bacteria into sewage to be inactivated with an initial carbon-nitrogen ratio less than 3.0, activating and expanding the heterotrophic bacteria for the first time; increasing the carbon-nitrogen ratio of the sewage to the range of 5.0-10.0, and activating and expanding the heterotrophic bacteria for the second time; washing and resuspending the activated and expanded heterotrophic bacteria to form a heterotrophic bacteria solution to be used; inoculating the heterotrophic bacteria solution into the sewage and reducing the carbon-nitrogen ratio of the sewage, so that the heterotrophic bacteria produce oxidative stress to inactivate the viruses.

[0023] The present application is based on the fact that microorganisms such as heterotrophic bacteria will activate oxidative stress pathways when they face conditions of nutrient deficiency, increase electron transfer inside and outside the cell, and increase the production of oxidative active species such as superoxide radicals; and oxidative active species such as superoxide radicals will attack the virus shell, thereby increasing the inactivation rate of viruses, especially enveloped viruses.

[0024] The present application first increases the carbon-nitrogen ratio of the sewage during the activation and expansion of the heterotrophic bacteria, so that the heterotrophic bacteria are in an eutrophic environment; and then reduces the carbon-nitrogen ratio of the sewage, so that the environment of the heterotrophic bacteria becomes oligotrophic, thereby causing the heterotrophic bacteria to produce oxidative stress to inactivate the viruses.

[0025] The method of the present application has good environmental friendliness, can provide practical support and new ideas for public health and ecological protection, and thus better cope with the challenges brought by viral infections.

[0026] The method of the present application is simple and efficient, and has a wide range of application scenarios.

[0027] Other features and advantages of the present application will be set forth in the following specification, and in part will be apparent from the description, or can be learned by practice of the present application. Other advantages of the present application will be realized and attained by the solution described hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0028] The accompanying drawings are included to provide a further understanding of the solution of the present application, and constitute a part of the specification, and are used together with the embodiments of the present application to explain the solution of the present application, and do not constitute a limitation to the solution of the present application.

[0029] Figure 1 The figure shows the comparison of the inactivation rate of Φ6 bacteriophage by the method for inactivating viruses in sewage provided in Example 1 of the present application and the inactivation rate of each control group;

[0030] Figure 2 The figure shows the comparison of the inactivation rate of MS2 bacteriophage by the method for inactivating viruses in sewage provided in Example 2 of the present application and the inactivation rate of each control group; and

[0031] Figure 3 The figure shows the comparison of the inactivation rate of Φ6 bacteriophage by the method for inactivating viruses in sewage provided in Example 3 of the present application and the inactivation rate of each control group. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present application more clear, the following text will make a detailed description of the embodiments of the present application. It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other at will.

[0033] In the following embodiments and control groups, the operations involved are carried out according to the conventional conditions or the conditions recommended by the manufacturer, if the conditions are not specified. The raw materials used are all products that can be obtained through market purchase, if the manufacturers and specifications are not specified.

[0034] Example 1

[0035] The present embodiment provides a method for inactivating viruses in sewage, which promotes the inactivation of Φ6 bacteriophage by Paracoccus denitrificans.

[0036] The sewage in the present embodiment can be composed of carbon source, nitrogen source and trace elements, in which sodium acetate is used as the carbon source, sodium nitrate is used as the nitrogen source, and the combination of 0.41 mM MgSO4, 19.6 μM Na2-EDTA, 9 μM FeCl3·6H2O, 0.1 μM MnCl2·4H2O, 0.8 μM Na2MoO4·2H2O, 0.8 μM CuCl2·2H2O and 2.5 μM ZnCl2 is used as the trace elements.

[0037] The method may include the following steps:

[0038] (1) The initial carbon-nitrogen ratio of the virus containing Φ6 bacteriophage ( ) was approximately 2.5:1. Phi 6 bacteriophage Wastewater from DSM21518, Leibniz Institute DSMZ was introduced into the first reaction tank. A buffer solution consisting of 10.66 mM NH4Cl, 17.93 mM KH2PO4, and 32.76 mM Na2HPO4·12H2O was added to adjust the pH of the wastewater to approximately 7. Denitrifying paracocci (…) were then added. Paracoccus Denitrificans The *Paracoccus denitrifyingans* strain (ATCC19367, Guangdong Provincial Microbial Culture Collection Center) underwent its first activation and expansion culture. The activation temperature was 37℃, the stirring rate was 300 rpm, and the activation time was 24 hours. After the first activation and expansion culture, the concentration (OD) of *Paracoccus denitrifyingans* in the wastewater was... 600 It is approximately 0.8-1.0.

[0039] (2) Wastewater from the first reaction tank was introduced into the second reaction tank, and sodium acetate was added as a carbon source to increase the carbon-to-nitrogen ratio of the wastewater in the second reaction tank to approximately 5:1, thus performing a second activation and expansion of *Paracoccus denitrifyingans*. The second activation and expansion was carried out under anoxic conditions at an activation temperature of 37°C, a stirring rate of 300 rpm, and an activation time of 24 hours. After the second activation and expansion, the concentration of *Paracoccus denitrifyingans* in the wastewater (OD) was... 600 It is approximately 0.8-1.0.

[0040] (3) Centrifuge the activated and expanded Paracoccus denitrified in the second reaction tank at 4°C and 5000g for 15 min, discard the supernatant, and resuspend it with PBS buffer. The concentration of the PBS buffer is 0.01M and the pH is 7.4±0.1. After centrifugation-washing-resuspending 3 times, use the last resuspended solution as the Paracoccus denitrified bacterial solution to be used.

[0041] (4) Place wastewater with a carbon-to-nitrogen ratio of approximately 1.25:1 in the third reaction tank, and inoculate the denitrifying Paracoccus bacteria solution into the third reaction tank at an inoculation concentration of OD. 600 The value was approximately 0.3; the third reaction tank was placed in an oxygen-deficient environment for virus inactivation reaction, with the temperature controlled at 37℃, the stirring rate at 300 rpm, and the inactivation time at 6 hours.

[0042] (5) Samples were taken at 0h and 6h of the reaction using a syringe. Sampling should ensure that the reactor is thoroughly mixed and in a near-homogeneous state. After sampling, the samples were filtered through a 0.22μm needle filter to remove denitrifying paracocci. The remaining liquid was immediately subjected to RNA extraction, reverse transcription, and qPCR to determine the inactivation rate of Φ6 phage before and after the reaction.

[0043] The formula for calculating the virus inactivation rate is as follows:

[0044] Virus inactivation rate = (initial virus copy number - final virus copy number) / final virus copy number x 100%; the test results are shown in Table 1. Figure 1

[0045] In addition, a control group 1 in which the carbon-nitrogen ratio of the sewage is maintained at about 5:1 for inactivation, a control group 2 in which no heterotrophic bacteria solution is added, and a control group 3 in which the heterotrophic bacteria solution is replaced with a pasteurized heterotrophic bacteria solution are also provided.

[0046] The virus inactivation rates of Example 1 and the control groups 1-3 are tested and calculated, and the results are shown in Table 2. Figure 1

[0047] As can be seen from Table 2, Figure 1 within 6 hours, the inactivation rate of Φ6 bacteriophage is 3.43 logs after the carbon-nitrogen ratio is reduced to 1.25:1, reaching an inactivation rate of more than 99.9%. In contrast, in the control group 1, the inactivation rate of Φ6 bacteriophage is 1.76 logs; in the control group 2, the inactivation rate of Φ6 bacteriophage is 0.51 logs; and in the control group 3, the inactivation rate of Φ6 bacteriophage is 0.48 logs. Therefore, by increasing and then decreasing the carbon-nitrogen ratio, the environment of the heterotrophic bacteria is changed from eutrophication to oligotrophy, thereby significantly improving the inactivation rate of viruses.

[0048] Example 2

[0049] The present embodiment provides a method for inactivating viruses in sewage, which promotes the inactivation of MS2 bacteriophage by Paracoccus denitrificans.

[0050] The method of the present embodiment differs from Example 1 in that the Φ6 bacteriophage in the sewage is replaced with MS2 bacteriophage (BNCC358039, Henan Beina Biological-Industrial Microbial Strain Engineering Technology Research Center), and the remaining conditions and steps are the same as those of Example 1. MS2 bacteriophage

[0051] In addition, a control group 4 in which the carbon-nitrogen ratio of the sewage is maintained at about 5:1 for inactivation, a control group 5 in which no heterotrophic bacteria solution is added, and a control group 6 in which the heterotrophic bacteria solution is replaced with a pasteurized heterotrophic bacteria solution are also provided.

[0052] The virus inactivation rates of Example 2 and the control groups 4-6 are tested and calculated, and the results are shown in Table 3. Figure 2

[0053] As can be seen from Table 3, Figure 2 ​​​​It can be seen that the method of first increasing and then decreasing the carbon-nitrogen ratio in this application can significantly increase the inactivation rate of MS2 bacteriophage from 0.31 log in the control group 4 to 0.8 log in this example 2.

[0054] Example 3

[0055] This embodiment provides a method for inactivating viruses in wastewater, which promotes the inactivation of Φ6 bacteriophage by Bacillus subtilis.

[0056] In this embodiment, the wastewater can be composed of a carbon source, a nitrogen source, and trace elements. Glucose is used as the carbon source, ammonium chloride is used as the nitrogen source, and a combination of 2mM MgSO4 and 0.1mM CaCl2 is used as the trace elements.

[0057] The method may include the following steps:

[0058] (1) The initial carbon-nitrogen ratio of the virus containing Φ6 bacteriophage ( ) was approximately 2.5:1. Phi 6 bacteriophage Wastewater from DSM21518, Leibniz Institute DSMZ was introduced into the first reaction tank. A buffer solution consisting of 10.66 mM NH4Cl, 17.93 mM KH2PO4, and 32.76 mM Na2HPO4·12H2O was added to adjust the pH of the wastewater to approximately 7. Bacillus subtilis (DSM21518, Leibniz Institute DSMZ) was then added. Bacillus subtilis (BNCC338006, Beina Biotechnology - Henan Provincial Industrial Microbial Strains Engineering Technology Research Center) conducted the first activation and expansion of Bacillus subtilis. The activation temperature was 37℃, the stirring rate was 300 rpm, and the activation time was 24 hours. After the first activation and expansion, the concentration (OD) of Bacillus subtilis in the wastewater was... 600 It is approximately 0.8-1.0.

[0059] (2) Wastewater from the first reaction tank was introduced into the second reaction tank, and glucose was added as a carbon source to increase the carbon-to-nitrogen ratio of the wastewater in the second reaction tank to approximately 5:1, for a second activation and expansion of Bacillus subtilis. The second activation and expansion was carried out under an aerobic environment at an activation temperature of 37°C, a stirring rate of 300 rpm, and an activation time of 24 hours. After the second activation and expansion, the concentration of Bacillus subtilis in the wastewater was OD 600 It is approximately 0.8-1.0.

[0060] (3) After separating the Bacillus subtilis in the second reaction tank from the sewage, the sewage is introduced into the third reaction tank; the activated and expanded Bacillus subtilis is centrifuged at 4°C and 5000g for 15 min, the supernatant is discarded, and the Bacillus subtilis is resuspended with PBS buffer, the concentration of the PBS buffer is 0.01M, and the pH is 7.4±0.1; after centrifugation-washing-resuspension for 3 times, the last resuspension is used as the Bacillus subtilis liquid to be used.

[0061] (4) The Bacillus subtilis liquid to be used is inoculated into the third reaction tank, the inoculation concentration OD 600 is about 0.3, and the carbon-nitrogen ratio in the third reaction tank is adjusted to about 1.25:1 (such as by adding ammonium chloride as a nitrogen source, dilution, etc.); the third reaction tank is placed in an aerobic environment for viral inactivation reaction, the temperature is controlled at 37°C, the stirring rate is 300 rpm, and the inactivation time is 6 hours.

[0062] (5) Sampling is performed at 0h and 6h of the reaction by a needle syringe. The sampling should ensure that the reactor is in a nearly homogeneous state in the fully mixed state. After sampling, the Bacillus subtilis in the sample is removed by a 0.22μm needle filter, and the remaining liquid is immediately subjected to RNA extraction, reverse transcription and qPCR processing to determine the inactivation rate of Φ6 bacteriophage before and after the reaction.

[0063] The calculation formula of the viral inactivation rate is as follows:

[0064] Viral inactivation rate = (initial virus copy number - final virus copy number) / final virus copy number x 100%; the test results are shown in Figure 3 .

[0065] In addition, a control group 7 is also provided, in which the carbon-nitrogen ratio of the sewage is maintained at about 5:1 for inactivation, a control group 8 in which no heterotrophic bacterial liquid is added, and a control group 9 in which the heterotrophic bacterial liquid is replaced by a heterotrophic bacterial liquid after pasteurization.

[0066] The viral inactivation rates of Example 3 and control groups 7-9 are tested and calculated, and the results are shown in Figure 3 .

[0067] From Figure 3 it can be seen that within 6 hours, after the carbon-nitrogen ratio is reduced to 1.25:1, the inactivation rate of Φ6 bacteriophage is increased from 0.61 log of the control group 7 to 1.04 log of the present example 3, and the inactivation rate is significantly improved.

[0068] Therefore, it can be seen that by adjusting the carbon-nitrogen ratio of the sewage to be inactivated, i.e. by increasing and then decreasing, the viral inactivation rate can be significantly improved.

[0069] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A method for inactivating viruses in sewage, characterized in that, The method comprises introducing heterotrophic bacteria into the sewage to be inactivated; increasing the carbon-nitrogen ratio of the sewage during the activation and cultivation of the heterotrophic bacteria; washing and resuspending the activated and cultivated heterotrophic bacteria to form a heterotrophic bacteria solution to be used; inoculating the heterotrophic bacteria solution into the sewage and reducing the carbon-nitrogen ratio of the sewage, so that the heterotrophic bacteria produce an oxidative stress response to inactivate the viruses; The activation and cultivation of the heterotrophic bacteria comprises first activation and cultivation of the heterotrophic bacteria in the sewage with an initial carbon-nitrogen ratio less than or equal to 3.0; and second activation and cultivation of the heterotrophic bacteria in the sewage with an increased carbon-nitrogen ratio in the range of 5.0-10.

0. The reduction of the carbon-nitrogen ratio of the sewage comprises reducing the carbon-nitrogen ratio of the sewage to the range of 0.2-1.5:

1.

2. The method of claim 1, wherein, The first activation and cultivation is performed for 6-30 hours; and / or The second activation and cultivation is performed for 6-30 hours; and / or The first and second activation and cultivation are both performed at a temperature in the range of 25-37°C.

3. The method of claim 1, wherein, The heterotrophic bacteria are selected from one or more of Paracoccus denitrificans (ATCC 19367) Paracoccus denitrificans ), Bacillus subtilis (ATCC 6051) Bacillus subtilis ), and Geobacter sulfurreducens (ATCC 51513) Geobacter sulfurreducens pca ).

4. The method of claim 1, wherein, The viruses are selected from one or more of Φ6 bacteriophage, MS2 bacteriophage, T4 bacteriophage, and T7 bacteriophage.

5. The method according to any one of claims 1-4, characterized in that, The sewage comprises a carbon source, a nitrogen source, and trace elements.

6. The method of claim 5, wherein, The carbon source is selected from one or more of sodium acetate, sodium citrate, glycerol, and glucose; and / or The nitrogen source is selected from one or more of sodium nitrate, peptone, and ammonium chloride; and / or The trace elements are selected from one or more of MgSO4, Na2-EDTA, FeCl3·6H2O, MnCl2·4H2O, Na2MoO4·2H2O, CuCl2·2H2O, CaCl2, and ZnCl2.

7. The method according to any one of claims 1-4, characterized in that, After activation and cultivation, the heterotrophic bacteria are washed and resuspended with a biological buffer solution.

8. The method of claim 7, wherein, The biological buffer solution is selected from at least one of Tris-HCl buffer solution, HEPES buffer solution, and PBS buffer solution.

9. The method of claim 8, wherein, The concentration of the biological buffer solution is 20-100 mM; and the pH of the biological buffer solution is 7.0-7.

6.

10. The method of claim 1, wherein, The inoculation concentration OD of the heterotrophic bacteria liquid into the sewage is 0.1-0.5 600 In the range of 0.1-0.5.

Images