Method for inactivating viruses in sewage

Abstract

The invention relates to a method for inactivating viruses in sewage. The method comprises the following steps: introducing heterotrophic bacteria into sewage to be inactivated; the carbon-nitrogen ratio of the sewage is increased in the process of activating and expanding the heterotrophic bacteria; washing and resuspending the activated and propagated heterotrophic bacteria to form a standby heterotrophic bacteria solution; the heterotrophic bacteria liquid is inoculated into the sewage, and the carbon nitrogen ratio of the sewage is reduced, so that the heterotrophic bacteria generate oxidative stress reaction to inactivate viruses. According to the method, the carbon nitrogen ratio is regulated and controlled, so that the heterotrophic bacteria are converted from a eutrophication environment to a hypotrophic environment, an oxidative stress pathway of the heterotrophic bacteria is activated, generation of oxidative active species such as superoxide radicals is increased, oxidative damage to virus shells or envelopes is caused, and virus inactivation is promoted. The method is simple and efficient, and has a wide application scene.

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 Art

[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 methods of virus inactivation include thermal inactivation, chemical inactivation, and physical inactivation. 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 of the invention

[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 process of activating and expanding the heterotrophic bacteria; washing and resuspending the activated and expanded heterotrophic bacteria to form a heterotrophic bacterial liquid to be used; inoculating the heterotrophic bacterial 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 the virus.

[0006] In the present application, the term "sewage" may also be referred to as wastewater, which generally includes industrial wastewater, discharged domestic sewage, agricultural wastewater, etc., but does not contain highly toxic or highly oxidizing chemicals, such as haloacetic acids, hydrogen peroxide, or its content does not affect the activity of microorganisms. In the present application, the initial carbon-nitrogen ratio of the sewage may be no greater than 3.0, such as in the range of 0.5-3.0.

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

[0008] In one embodiment, activating and expanding the heterotrophic bacteria may include: performing a first activation and expansion on the heterotrophic bacteria in the sewage having an initial carbon-nitrogen ratio less than or equal to 3.0; and performing a second activation and expansion on the heterotrophic bacteria in the sewage having a carbon-nitrogen ratio increased to a range of 5.0-10.0.

[0009] In one embodiment, the first activation and expansion time is 6-30 hours.

[0010] In one embodiment, the second activation expansion time is 6-30h.

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

[0012] In one embodiment, reducing the carbon-nitrogen ratio of the sewage includes reducing the carbon-nitrogen ratio of the sewage to a range of 0.2-1.5:1.

[0013] In one embodiment, the heterotrophic bacteria are selected from Paracoccus denitrificans ( Paracoccus denitrificans )、Bacillus subtilis( Bacillus subtilis ) and Geobacter sulfurreducens ( Geobacter sulfurreducens pca )

[0014] In one embodiment, the virus is selected from Φ6 bacteriophage ( Φ6 bacteriophage )、MS2 bacteriophage( MS2 Bacteriophage )、T4 phage( T4 bacteriophage ) and T7 phage ( T4 bacteriophage )

[0015] In one embodiment, the sewage includes a carbon source, a nitrogen source and trace elements.

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

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

[0018] In one embodiment, the trace element is selected from one or more of MgSO4, Na2-EDTA, FeCl3·6H2O, MnCl2·4H2O, Na2MoO4·2H2O, CuCl2·2H2O, CaCl2 and ZnCl2. Alternatively, the trace element is selected from two or more of MgSO4, Na2-EDTA, FeCl3·6H2O, MnCl2·4H2O, Na2MoO4·2H2O, CuCl2·2H2O, CaCl2 and ZnCl2.

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

[0020] In one embodiment, after activation and expansion, 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 one embodiment, the inoculation concentration OD of the heterotrophic bacterial solution inoculated into the sewage is 600 In the range of 0.1-0.5.

[0022] On the other hand, 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 of less than 3.0, and performing a first activation and expansion culture on the heterotrophic bacteria; increasing the carbon-nitrogen ratio of the sewage to within the range of 5.0-10.0, and performing a second activation and expansion culture on the heterotrophic bacteria; washing and resuspending the heterotrophic bacteria after activation and expansion to form a heterotrophic bacterial liquid to be used; inoculating the heterotrophic bacterial 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 the virus.

[0023] The present application is based on the fact that when microorganisms such as heterotrophic bacteria face conditions of nutrient deficiency, they will activate oxidative stress pathways, increase electron transfer inside and outside the cells, and increase the production of oxidative active species such as superoxide free radicals; and oxidative active species such as superoxide free 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 process of activating and expanding the heterotrophic bacteria, so that the heterotrophic bacteria are in a eutrophic environment; and then reduces the carbon-nitrogen ratio of the sewage, so that the environment in which the heterotrophic bacteria are located becomes a oligotrophic environment, thereby causing the heterotrophic bacteria to produce an oxidative stress response to inactivate the virus.

[0025] The method of the present application is environmentally friendly and can provide practical support and new ideas for public health and ecological protection, thereby better responding to 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 described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical 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; 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 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

[0030] In order to make the purpose, technical solution and advantages of the present application more clear, the embodiments of the present application are described in detail below. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other arbitrarily without conflict.

[0031] In the following examples and control groups, the operations involved were carried out under conventional conditions or conditions recommended by the manufacturer if the conditions were not specified. The raw materials used were commercially available if the manufacturers and specifications were not specified.

[0032] Example 1 This embodiment provides a method for inactivating viruses in sewage, which promotes the inactivation of Φ6 bacteriophage by Paracoccus denitrificans.

[0033] The sewage in this embodiment can be composed of a carbon source, a nitrogen source and trace elements, wherein sodium acetate is used as the carbon source, sodium nitrate is used as the nitrogen source, and a combination of 0.41mM MgSO4, 19.6μM Na2-EDTA, 9μM FeCl3·6H2O, 0.1μM MnCl2·4H2O, 0.8μM Na2MoO4·2H2O, 0.8μM CuCl2·2H2O and 2.5μM ZnCl2 is used as trace elements.

[0034] The method may include the following steps: (1) The initial carbon-nitrogen ratio of about 2.5:1 containing Φ6 bacteriophage virus ( Φ6 bacteriophage , DSM21518, Leibniz Institute DSMZ) into the first reaction tank, add a buffer solution consisting of 10.66mM NH4Cl, 17.93mM KH2PO4, 32.76mM Na2HPO4·12H2O to adjust the pH of the sewage to about 7, add denitrifying Paracoccus ( Paracoccus Denitrificans , ATCC19367, Guangdong Provincial Microbial Culture Collection Center), the first activation and expansion of denitrifying Paracoccus was carried out. The activation temperature was 37°C, the stirring rate was 300rpm, and the activation time was 24 hours. After the first activation and expansion, the concentration of denitrifying Paracoccus in the sewage was OD 600 About 0.8-1.0.

[0035] (2) The sewage in the first reaction tank was introduced into the second reaction tank, and sodium acetate was added as a carbon source to increase the carbon-nitrogen ratio of the sewage in the second reaction tank to about 5:1, and the denitrifying Paracoccus was activated and expanded for the second time. The second activation and expansion was carried out in an anaerobic environment, with 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 denitrifying Paracoccus in the sewage was OD 600 About 0.8-1.0.

[0036] (3) The activated and expanded Paracoccus denitrifyingii in the second reaction tank was centrifuged at 4°C and 5000 g for 15 min, the supernatant was discarded, and the culture was resuspended in PBS buffer, wherein the concentration of the PBS buffer was 0.01 M and the pH was 7.4±0.1; after centrifugation-washing-resuspension 3 times, the last resuspension was used as the Paracoccus denitrifyingii bacterial liquid to be used.

[0037] (4) Place sewage with a carbon-nitrogen ratio of about 1.25:1 in the third reaction tank, and inoculate the denitrifying Paracoccus bacteria solution to be used into the third reaction tank at an inoculation concentration of OD 600 The third reaction pool was placed in an anaerobic environment for virus inactivation reaction, the temperature was controlled at 37°C, the stirring rate was 300rpm, and the inactivation time was 6 hours.

[0038] (5) Samples were taken at 0h and 6h of the reaction using a syringe needle. The samples should be mixed thoroughly in the reactor to ensure that the reactor was nearly homogeneous. After sampling, the denitrifying Paracoccus in the sample was removed through a 0.22μm syringe filter, and 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.

[0039] The formula for calculating the virus inactivation rate is as follows: Virus inactivation rate = (initial virus copy number - final virus copy number) / final virus copy number × 100%; test results are as follows Figure 1 shown.

[0040] 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 bacterial solution is added, and a control group 3 in which the heterotrophic bacterial solution is replaced with a pasteurized heterotrophic bacterial solution are provided.

[0041] The virus inactivation rate was tested and calculated for Example 1 and Control Groups 1-3. The results are as follows: Figure 1 shown.

[0042] Depend on Figure 1 It can be seen that within 6 hours, after reducing the carbon-nitrogen ratio to 1.25:1, the inactivation rate of Φ6 bacteriophage was 3.43 logs, reaching an inactivation rate of more than 99.9%. In contrast, in control group 1, the inactivation rate of Φ6 bacteriophage was 1.76 logs; in control group 2, the inactivation rate of Φ6 bacteriophage was 0.51 logs; in control group 3, the inactivation rate of Φ6 bacteriophage was 0.48 logs. Therefore, the present application changes the environment of heterotrophic bacteria from eutrophication to oligotrophication by first increasing and then reducing the carbon-nitrogen ratio, thereby significantly improving the inactivation rate of viruses.

[0043] Example 2 This embodiment provides a method for inactivating viruses in sewage, which promotes the inactivation of MS2 bacteriophage by Paracoccus denitrificans.

[0044] The method of this embodiment is different from that of embodiment 1 in that the Φ6 bacteriophage in the sewage is replaced with MS2 bacteriophage ( MS2 bacteriophage , BNCC358039, Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center), the remaining conditions and steps are the same as Example 1.

[0045] In addition, a control group 4 in which the carbon-nitrogen ratio of the sewage was maintained at about 5:1 for inactivation, a control group 5 in which no heterotrophic bacterial solution was added, and a control group 6 in which the heterotrophic bacterial solution was replaced with a pasteurized heterotrophic bacterial solution were provided.

[0046] The virus inactivation rate was tested and calculated for Example 2 and control groups 4-6. The results are as follows Figure 2 shown.

[0047] Depend on Figure 2 It can be seen that the method of first increasing and then reducing the carbon-nitrogen ratio of the present 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.

[0048] Example 3 This embodiment provides a method for inactivating viruses in sewage, which promotes the inactivation of Φ6 bacteriophage by Bacillus subtilis.

[0049] The sewage in this embodiment may be composed of a carbon source, a nitrogen source and trace elements, wherein glucose is used as the carbon source, ammonium chloride is used as the nitrogen source, and a combination of 2 mM MgSO 4 and 0.1 mM CaCl 2 is used as the trace element.

[0050] The method may include the following steps: (1) The initial carbon-nitrogen ratio of about 2.5:1 containing Φ6 bacteriophage virus ( Φ6 bacteriophage , DSM21518, Leibniz Institute DSMZ) sewage was introduced into the first reaction tank, and a buffer solution consisting of 10.66mM NH4Cl, 17.93mM KH2PO4, and 32.76mM Na2HPO4·12H2O was added to adjust the pH of the sewage to about 7, and Bacillus subtilis ( Bacillus subtilis , BNCC338006, Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center), the first activation and expansion of Bacillus subtilis. The activation temperature was 37°C, the stirring rate was 300rpm, and the activation time was 24 hours. After the first activation and expansion, the concentration of Bacillus subtilis in the sewage was OD 600 About 0.8-1.0.

[0051] (2) The sewage in the first reaction tank was introduced into the second reaction tank, and glucose was added as a carbon source to increase the carbon-nitrogen ratio of the sewage in the second reaction tank to about 5:1, and the Bacillus subtilis was activated and expanded for the second time. The second activation and expansion was carried out in an aerobic environment, with 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 sewage was OD 600 About 0.8-1.0.

[0052] (3) After separating the Bacillus subtilis from the sewage in the second reaction tank, 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 culture is resuspended with PBS buffer, wherein the concentration of the PBS buffer is 0.01 M and the pH is 7.4±0.1; after centrifugation-washing-resuspension 3 times, the last resuspension is used as the Bacillus subtilis bacterial liquid to be used.

[0053] (4) Inoculate the Bacillus subtilis solution to be used into the third reaction tank, with an inoculation concentration of OD 600The carbon-nitrogen ratio is about 0.3, and the carbon-nitrogen ratio is adjusted to about 1.25:1 in the third reaction tank (e.g., by adding ammonium chloride as a nitrogen source, diluting, etc.); the third reaction tank is placed in an aerobic environment for virus inactivation reaction, the temperature is controlled at 37°C, the stirring rate is 300rpm, and the inactivation time is 6 hours.

[0054] (5) Samples were taken at 0h and 6h of the reaction using a syringe needle. The samples should be mixed thoroughly in the reactor to ensure that the reactor was nearly homogeneous. After sampling, the Bacillus subtilis in the sample was removed through a 0.22μm syringe filter, and 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.

[0055] The formula for calculating the virus inactivation rate is as follows: Virus inactivation rate = (initial virus copy number - final virus copy number) / final virus copy number × 100%; test results are as follows Figure 3 shown.

[0056] In addition, a control group 7 in which the carbon-nitrogen ratio of the sewage was maintained at about 5:1 for inactivation, a control group 8 in which no heterotrophic bacterial solution was added, and a control group 9 in which the heterotrophic bacterial solution was replaced with a pasteurized heterotrophic bacterial solution were provided.

[0057] The virus inactivation rate was tested and calculated for Example 3 and control groups 7-9. The results are as follows: Figure 3 shown.

[0058] Depend on Figure 3 It can be seen that within 6 hours, after reducing the carbon-nitrogen ratio to 1.25:1, the inactivation rate of Φ6 bacteriophage increased from 0.61 log in the control group 7 to 1.04 log in this example 3, and the inactivation rate was significantly improved.

[0059] It can be seen that the present application can achieve a significant improvement in the virus inactivation rate by regulating the carbon-nitrogen ratio of the sewage to be inactivated, that is, by first increasing and then decreasing it.

[0060] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Those skilled in the art can change, modify, replace and modify the above embodiments within 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 process of activating and expanding the heterotrophic bacteria; washing and resuspending the activated and expanded heterotrophic bacteria to form a heterotrophic bacterial liquid to be used; inoculating the heterotrophic bacterial 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 the virus.

2. The method according to claim 1, characterized in that Activating and expanding the heterotrophic bacteria includes performing a first activation and expansion on the heterotrophic bacteria in the sewage with an initial carbon-nitrogen ratio of less than or equal to 3.0; and performing a second activation and expansion on the heterotrophic bacteria in the sewage with a carbon-nitrogen ratio increased to a range of 5.0-10.

0.

3. The method according to claim 2, characterized in that The first activation and expansion time is 6-30h; and / or, The second activation and expansion time is 6-30h; and / or, The first activation expansion and the second activation expansion are both carried out in the temperature range of 25-37°C.

4. The method according to claim 1, characterized in that Reducing the carbon-nitrogen ratio of the sewage includes reducing the carbon-nitrogen ratio of the sewage to a range of 0.2-1.5:

1.

5. The method according to claim 1, characterized in that The heterotrophic bacteria are selected from Paracoccus denitrificans ( Paracoccus denitrificans )、Bacillus subtilis( Bacillus subtilis ) and Geobacter sulfurreducens ( Geobacter sulfurreducens pca ) 6. The method according to claim 1, characterized in that The virus is selected from one or more of Φ6 phage, MS2 phage, T4 phage and T7 phage.

7. The method according to any one of claims 1 to 6, characterized in that The sewage comprises a carbon source, a nitrogen source and trace elements.

8. The method according to claim 7, characterized in that 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.

9. The method according to any one of claims 1 to 6, characterized in that: After activation and expansion, 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.

10. The method according to claim 1, characterized in that The inoculation concentration OD of the heterotrophic bacterial solution inoculated into the sewage 600 In the range of 0.1-0.5.

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