Phage, phage preparation and application of phage preparation in reduction of nitrous oxide emission
By using Shigella Fukura phage B003 preparation to target the reduction of bacteria produced by nitrous oxide in composting products, the problem of nitrous oxide emissions during compost products is solved, significant reductions in nitrous oxide emissions are achieved, and environmental protection of compost products is improved.
Patent Information
- Application Number
- CN202411752296.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-03-07
AI Technical Summary
Compost products will emit a large amount of nitrous oxide during storage, resulting in enhanced greenhouse effect and damage to the ozone layer. The existing technology is difficult to effectively solve this problem.
The phage preparation prepared by Shigella Fukura phage B003 is used, and the phage preparations are sprayed in a composting product, and the lytic activity of the phage is used to target and reduce the nitrous oxide-producing bacteria.
Effectively reduce the emission of nitrous oxide during compost products storage, reduce the risk of greenhouse gas emissions, and improve the environmental protection of compost products.
Smart Images
Figure CN119570746A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and more specifically, to a bacteriophage, a bacteriophage preparation and application thereof in reducing nitrous oxide emissions. Background Art
[0002] Compost products are rich in organic matter and nutrients required by plants and are good soil conditioners. Relevant studies have shown that compost products emit a large amount of nitrous oxide during storage. Nitrous oxide is a powerful greenhouse gas with a greenhouse effect 300 times higher than carbon dioxide. It can stay in the atmosphere for a long time and destroy the ozone layer. If large-volume compost products ignore nitrous oxide emissions and are stored for a long time, it may disrupt the global nitrogen cycle and accelerate global warming. Therefore, it is of great significance to develop a treatment method to reduce nitrous oxide emissions in compost products.
[0003] Bacteriophage (phage) is a type of virus that infects prokaryotes such as bacteria and is widely distributed in artificial and natural environments such as digestion reactors, compost, soil, and water. Bacteriophages can specifically infect and lyse bacteria, and will not infect other beneficial bacteria, causing no harm to humans, animals, and plants. Therefore, it is of great significance to use bacteriophages to target and lyse nitrous oxide-emitting bacteria, thereby reducing nitrous oxide emissions. Summary of the invention
[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a bacteriophage, a bacteriophage preparation and its application in reducing nitrous oxide emissions.
[0005] The first aspect of the present invention provides a bacteriophage, which is named Shigella flexneri phage B003, deposited in the China Center for Type Culture Collection on July 1, 2024, located in Wuhan, China, with a deposit number of CCTCC NO: M 20241434.
[0006] Furthermore, under electron microscopic observation, the phage B003 is a typical tadpole-shaped muscle-tailed phage, whose morphology includes a head and a tail. The head has a diameter of 80nm, and the tail has a contractile tail sheath and tail filaments. The tail is 115nm long.
[0007] Furthermore, bacteriophage B003 has lytic activity against Shigella flexneri at 4-75°C and pH 2.0-13.0.
[0008] Furthermore, the optimal infection multiplicity of phage B003 was 10.
[0009] Furthermore, the incubation period of bacteriophage B003 is 10 minutes, the outbreak period is 30 minutes, and it enters the plateau period after 40 minutes.
[0010] Furthermore, the bacteriophage B003 preservation method is to mix the bacteriophage lysate with 50% glycerol in a ratio of 1:1, and then store it at -20°C or -80°C.
[0011] Furthermore, Shigella flexneri produces nitrous oxide in the compost product.
[0012] The second aspect of the present invention provides a use of Shigella flexneri bacteriophage B003 in reducing nitrous oxide emissions.
[0013] The third aspect of the present invention provides a phage preparation, comprising a Shigella flexneri phage B003.
[0014] In some embodiments, the content of active Shigella flexneri phage B003 in the preparation is 10 8 ~10 10 PFU / mL.
[0015] In some embodiments, the preparation further comprises a pharmaceutically acceptable excipient, wherein the excipient comprises one or more of a stabilizer, a dispersant, a solvent or a filler.
[0016] In some embodiments, the dosage form of the preparation is at least one of a solution, a powder, a gel, a granule, or a lyophilized preparation.
[0017] The third aspect of the present invention provides the use of the above-mentioned bacteriophage preparation in reducing nitrous oxide emissions during the storage process of compost products.
[0018] A fourth aspect of the present invention provides a method for reducing nitrous oxide emissions during storage of compost products, the method comprising uniformly spraying the above-mentioned bacteriophage preparation into the compost product at a volume mass fraction ratio of 0.1 to 1%.
[0019] In some embodiments, the compost product is a product of livestock and poultry manure or agricultural product straw after 35 days of composting and fermentation. The compost product has a moisture content of 30%-40%, a pH value of 6.9-7.2, and an organic matter content of 40%-60%.
[0020] In some embodiments, the compost product is stored at a temperature of 15-35°C.
[0021] The beneficial effects of the present invention are as follows: the Shigella flexneri phage B003 of the present invention can adapt to the extreme environment in composting, maintain activity under the conditions of 3≤pH≤13, 4≤T≤75°C, and efficiently lyse bacteria that produce nitrous oxide, and can be propagated in large quantities. The phage preparation prepared by the phage B003 can be inoculated into compost products of agricultural product waste or livestock and poultry manure according to actual production needs, so as to achieve long-term reduction of nitrous oxide emissions during the storage of compost products, improve the environmental protection of compost products, and have broad practical value and environmental significance. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the plaque image of Shigella flexneri phage B003 of the present invention.
[0023] Figure 2 This is a diagram of the complete genome sequence analysis of the Shigella flexneri phage B003 of the present invention.
[0024] Figure 3 This is an electron microscope characterization image of the Shigella flexneri phage B003 of the present invention.
[0025] Figure 4 This is a characterization diagram of the infection multiplicity of the Shigella flexneri phage B003 of the present invention.
[0026] Figure 5 This is a characterization diagram of the one-step growth curve of the Shigella flexneri phage B003 of the present invention.
[0027] Figure 6 This is a thermal stability characterization diagram of the Shigella flexneri phage B003 of the present invention.
[0028] Figure 7 This is a characterization diagram of the acid-base sensitivity of the Shigella flexneri phage B003 of the present invention. DETAILED DESCRIPTION
[0029] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.
[0030] Test materials
[0031] Example 1 Isolation of host Shigella flexneri and monitoring of its nitrous oxide production
[0032] 1.1 Isolation of Shigella flexneri
[0033] Cow dung from a farm in Fuzhou City, Fujian Province was composted at high temperature. 2 g of mature compost sample was added into 18 mL of PBS buffer and shaken in a shaker at 37 °C and 220 rpm / min for 1 h. Then it was allowed to stand for 30 min. The supernatant was filtered through a 0.45 μm sterile filter and diluted to 10 -5 The filtrate was spread on a solid LB plate and cultured at 37°C for 48 hours. A single colony with a single color, smooth surface, uniform shape and neat edges was streaked on the solid LB plate and cultured at 37°C for 24 hours. The above steps were repeated 3 times to purify the host bacteria, and the bacterial solution of the host bacteria was subjected to 16s RNA molecular identification.
[0034] 1.2 Monitoring of nitrous oxide production
[0035] The purified host bacteria were inoculated into anaerobic bottles and measured using GC-MS. The bacteria whose nitrous oxide production was 500 times greater than the nitrous oxide content in the air were selected as Shigella flexneri.
[0036] Example 2 Isolation and Identification of Shigella flexneri Phage B003
[0037] 2.1 Soil sample processing
[0038] 2 mL of the supernatant of the compost extract in 1.1 of Example 1 was taken and centrifuged at 4°C, 12000 r / min, 3 min, and the supernatant was filtered (0.22 μm water filter) to prepare a sterile phage suspension;
[0039] 2.2 Preparation of host bacterial culture
[0040] The nitrous oxide-producing bacterium Shigella flexneri was cultured in LB liquid medium at 37° C. and 220 rpm / min shaking for 6 hours to reach a logarithmic growth phase, thereby preparing a host bacterial solution;
[0041] 2.3 Isolation, purification and titer determination of bacteriophage B003
[0042] Take 100 μL of phage suspension and 100 μL of host bacterial liquid and add them to 5 mL of liquid LB medium, culture at 37°C overnight to obtain co-culture liquid. Take 2 mL of co-culture liquid, then centrifuge it at 12000 r / min and 4°C for 3 minutes, and then filter the supernatant (0.22 μm water filter) to obtain a high-titer phage suspension.
[0043] The high titer phage suspension was diluted in series (10 -1 ~10 -8), take 10μL of each gradient phage suspension and drop it on the solidified LB plate containing bacteria, culture it at 37℃ overnight, record the number of plaques that appear in the highest dilution gradient, and calculate the titer. Phage titer (PFU / mL) = number of plaques × 100 × dilution multiple. Take a single plaque and inoculate it in 5mL liquid LB containing 100μL host bacterial solution, culture it at 37℃ overnight, and centrifuge to obtain a high-concentration phage suspension. Repeat the above operation 3 times to obtain purified high-titer Shigella flexneri phage. The plaques are as follows: Figure 1 shown.
[0044] 2.4 Whole-genome sequencing
[0045] The whole genome of the phage was sequenced and compared with the NCBI Refseq virus database. The three Escherichia coli phages with the highest homology were selected, with coverage above 90% and identity above 95%, and their genomes were compared for colinearity. The results are shown in Figure 2 .
[0046] like Figure 2 As shown, the local genes of phage B003 are different from those of the other three phages, the local gene arrangement is obviously different, and it cannot infect Escherichia coli. Therefore, phage B003 is a new phage, named Shigella flexneri phage B003.
[0047] Example 3 Electron microscopic observation of Shigella flexneri phage B003
[0048] Take 20 μL of Shigella flexneri phage B003 lysate onto the copper mesh, let it stand for 5 minutes, remove the excess co-culture solution with filter paper, and dry for 1 minute. Add 1 drop of 1% phosphotungstic acid (PTA) to the copper mesh, let it stand at room temperature for 10 minutes, and observe the phage morphology under a transmission electron microscope after the sample is dry.
[0049] Phage morphology Figure 3 As shown, the head of the phage B003 is about 80 nm in diameter, and the tail is 115 nm in length with a contractile tail sheath and tail filaments. It is a typical tadpole-shaped muscle-tailed phage.
[0050] Example 4 Determination of the Optimal Multiplicity of Infection (MOI) of Shigella flexneri Phage B003
[0051] The isolated host bacteria were cultured to the logarithmic growth phase (10 8 CFU / mL), take 100 μL of different gradient titers (10 4 ~10 9The phage B003 suspension with 100 μL of bacterial suspension at MOI of 0.01, 0.1, 1, 10, and 100 was uniformly mixed in 5 mL of liquid LB, and then cultured at 37°C and 220 rpm for 12 h, and then centrifuged at 12000 rpm for 3 min. The supernatant was collected and filtered through a 0.22 μm sterile filter. The filtrate was diluted gradiently (10 -1 ~10 -8 ), 10 μL of each was dropped onto the LB solid plate coagulated with the host bacteria, cultured at 37°C overnight, the number of plaques was counted, and the phage titer was calculated. This was repeated three times, and the infection multiplicity with the highest titer was the optimal infection multiplicity. The characterization results were statistically reported in Figure 4 As shown. Figure 4 It can be seen that the optimal infection multiplicity of bacteriophage B003 is 10.
[0052] Example 5 One-step growth curve determination of Shigella flexneri phage B003
[0053] Under the condition of optimal infection multiplicity of 10, the host bacteria (10 8 CFU / mL) and phage suspension were added to liquid LB with a total volume of 20 mL. The suspension was cultured at 37°C for 15 min, then centrifuged at 12000 r / min, 4°C for 3 min, the supernatant was discarded, an appropriate amount of liquid LB was added, the precipitate was washed repeatedly by blowing, and the suspension was centrifuged at 12000 r / min, 4°C for 3 min, the supernatant was discarded, and repeated 3 times. 20 mL of liquid LB was added and cultured at 37°C with shaking at 170 rpm / min. Samples were taken every 5 min in the first 30 min, and every 10 min from 30 min to 150 min. Each time, 100 μL was taken and added to 900 μL of sterile water. The suspension was then centrifuged at 12000 r / min for 3 min, filtered, and the phage titer was measured. This was repeated 3 times. A one-step growth curve was drawn with the sampling time as the horizontal axis and the logarithm of the phage titer as the vertical axis, as shown in Figure 3. Figure 5 .
[0054] Depend on Figure 5 As can be seen, within 0-10 minutes, the titer did not change significantly, so the incubation period of phage B003 was about 10 minutes; within 10-40 minutes, the phage titer increased sharply, indicating that the outbreak period of phage B003 was about 30 minutes, and after 40 minutes, the titer tended to stabilize and entered the plateau period. This shows that phage B003 is a virulent phage that can infect and lyse host bacteria in a relatively short period of time and can proliferate in large quantities in a short period of time.
[0055] Example 6 Determination of thermal stability of Shigella flexneri phage B003
[0056] Take 1 mL of phage suspension (109 In 8 sterile centrifuge tubes, add (10⁶ PFU / mL), and incubate in a constant temperature water bath at 4°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 75°C for 2 h. Then, dilute it 10-fold with sterile water, and measure the phage titer by the spot method.
[0057] The results are as Figure 6 shown. In the range of 4 - 60°C, the phage has relatively stable lysis activity against the host bacteria. When the temperature is greater than 60°C, the phage activity drops sharply. However, at 70°C, the phage is not completely inactivated and still has a certain lysis activity. This indicates that the phage B003 has a wide temperature growth range and strong tolerance to temperature.
[0058] Example 7: Determination of the acid-base stability of Shigella flexneri phage B003
[0059] Respectively take 100 μL of the phage suspension (10⁶ 9 PFU / mL) and add it to 900 μL of sterile liquid LB with pH values of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. Incubate in a constant temperature water bath at 37°C for 2 h. Then, dilute it 10-fold with sterile water, and measure the phage titer by the spot method. The results are shown in Figure 7 .
[0060] As Figure 7 shown, when 3 < pH < 13, the phage B003 still maintains a high lysis activity. Under strong alkaline conditions (pH = 13), it is not completely inactivated and still has a certain lysis activity, indicating that the phage B003 has a certain tolerance to acids and bases, especially to bases.
[0061] Example 8: Preparation of phage preparation
[0062] Expand the culture of the host Shigella flexneri, and control the concentration of the host bacteria to be 10⁸ 8 ~10⁹ 9 CFU / mL. Inoculate the phage B003 into the host bacterial solution, and prepare a phage preparation through a 0.22 μm aqueous filter head, controlling the activity of phage B003 in the preparation to be 10⁷ 8 ~10⁸ 10 PFU / mL.
[0063] Example 9: Test on the reduction of nitrous oxide emissions in compost products by phage preparation
[0064] Treatment group 1:
[0065] 80L of livestock and poultry manure compost product was uniformly inoculated with the bacteriophage preparation prepared in Example 8 at a volume fraction of 0.1%, and the emission of nitrous oxide in the compost product was detected by a gas collection device and gas chromatography-mass spectrometry (GC-MS) on the first, seventh, fifteenth and thirtieth day of inoculation. During this process, the compost was not turned to simulate the storage of conventional compost products.
[0066] Control group 1:
[0067] Same as treatment group 1, except that the phage preparation added to the compost product was replaced with the same volume of sterile water.
[0068] Treatment Group 2:
[0069] 80L of agricultural straw compost was uniformly inoculated with the bacteriophage preparation prepared in Example 8 at a volume fraction of 0.1%, and the emission of nitrous oxide in the compost was detected by a gas collection device and gas chromatography-mass spectrometry (GC-MS) on the first, seventh, fifteenth and thirtieth day of inoculation. During this process, the compost was not turned to simulate the storage of conventional compost products.
[0070] Control group 2:
[0071] Same as treatment group 2, except that the phage preparation added to the compost product was replaced with the same volume of sterile water.
[0072] The specific results are shown in Table 1.
[0073] Table 1 Determination of nitrous oxide emissions in compost products
[0074]
[0075] As shown in Table 1, the results of treatment group 1 and control group 1 show that inoculation of bacteriophage B003 preparation in livestock and poultry manure compost products can effectively reduce nitrous oxide emissions, and the emission on the 30th day was reduced by 65%. The results of treatment group 2 and control group 2 show that inoculation of bacteriophage B003 preparation in agricultural straw compost products can effectively reduce nitrous oxide emissions, and the emission on the 30th day was reduced by 66%. It shows that the Shigella flexneri phage B003 and preparation prepared by the present invention have a significant effect on reducing nitrous oxide emissions during the storage of compost products.
[0076] The bacteriophage preparation for reducing nitrous oxide emission in compost products and the application method thereof of the present invention have the advantages of simple operation and significant reduction effect, and are suitable for composting of various organic wastes. By inoculating the Shigella flexneri bacteriophage B003 preparation, nitrous oxide emission during storage of compost products is reduced, the risk of greenhouse gas emission from compost products is reduced, and the safety and environmental protection of composting are improved, so that the present invention has broad application prospects in the field of composting.
[0077] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.
Claims
1. A bacteriophage, characterized in that: The phage was named Shigella flexneri phage B003, and was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number being CCTCC NO: M 20241434.
2. Use of a bacteriophage as claimed in claim 1 in reducing nitrous oxide emissions.
3. A bacteriophage preparation, characterized in that: It comprises the Shigella flexneri phage B003 as described in claim 1.
4. A bacteriophage preparation according to claim 3, characterized in that: The content of active Shigella flexneri bacteriophage B003 in the preparation is 10 8 ~10 10 PFU / mL.
5. A bacteriophage preparation according to claim 3, characterized in that: The preparation further comprises pharmaceutically acceptable excipients, which include one or more of a stabilizer, a dispersant, a solvent or a filler.
6. A bacteriophage preparation according to claim 3, characterized in that: The dosage form of the preparation is at least one of a solution, a powder, a gel, a granule or a lyophilized agent.
7. Use of a bacteriophage preparation according to any one of claims 3 to 6 for reducing nitrous oxide emissions during storage of compost products.
8. A method for reducing nitrous oxide emissions during storage of compost products, characterized in that: The method comprises spraying the bacteriophage preparation according to any one of claims 3 to 6 uniformly into the compost product at a volume mass fraction of 0.1 to 1%.
9. The method according to claim 8, characterized in that The compost product is a product of livestock and poultry manure or agricultural product straw after 35 days of composting and fermentation. The water content of the compost product is 30%-40%, the pH value is 6.9-7.2, and the organic matter content is 40%-60%.
10. The method according to claim 8, wherein the storage temperature of the compost product is 15-35°C.