Method for activating prophage based on low-voltage electrochemical technology
By activating prophages using a low-voltage electrochemical method, the problems of resistance development in host bacteria and chemical activation contamination have been solved. This approach achieves efficient and environmentally friendly phage activation and organic matter degradation, simplifies operation, and reduces energy consumption.
Patent Information
- Application Number
- CN202510876923.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-11-07
Smart Images
Figure CN120905159A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of environmental protection, and in particular to a method for activating a prophage based on low-voltage electrochemical technology. BACKGROUND
[0002] Phages are viruses that infect bacteria. In sewage treatment plants, the concentration of phages reaches 10 8 ~ 10 9 VLPs / ml, which is higher than in other aquatic systems. Phages directly affect prokaryotic communities through lytic infection and lysogenic conversion of their hosts. After lytic infection, phages reproduce at the expense of their hosts, using host resources to produce offspring, and regulate host bacterial density. After lysogenic infection, phages integrate their DNA into the host genome, forming prophages. Prophages remain dormant and replicate with host DNA, but under certain conditions, they can be activated and enter the lytic cycle. Studies have shown that about 20-40% of bacterial deaths are caused by phage lysis.
[0003] There have been many studies on using lytic phages to regulate the performance of biological treatment in sewage plants, such as controlling sludge bulking and membrane biological fouling. Due to the specificity of phage hosts, specific phages need to be pre-screened, and bacteria will develop resistance after long-term operation, reducing treatment effectiveness. Activating prophages into the lytic cycle through environmental stimuli has the advantage of universal occurrence and can produce long-term effective control results. Environmental stimuli include the addition of agents such as mitomycin, heavy metals, and antibiotics, but inevitably cause secondary environmental pollution.
[0004] Electrochemistry is an environmentally friendly technology with great development potential, with the advantages of small footprint, simple reactor, high degree of automation, and low investment cost. Active substances such as hydroxyl radicals (·OH) generated on the anode surface can oxidize and degrade pollutants in water and damage the structure and function of bacteria, causing bacterial damage and triggering the SOS response, which activates prophages. There is currently no research on activating prophages using low-voltage electrolysis. SUMMARY
[0005] To overcome the defects of the prior art, the present application provides a method for activating prophages based on low-voltage electrochemical technology, which activates prophages by efficiently generating active radicals, has the characteristics of simple operation, low energy consumption, and environmental friendliness.
[0006] To achieve the above purpose, the technical solution adopted by the present application is:
[0007] A method for activating prophages based on low-voltage electrochemical technology, comprising the following steps:
[0008] The low-voltage electrolysis is carried out on the bacterial electrolyte containing the original bacteriophage by using a double electrode system, and the electrolyzed solution is cultured in a culture solution, and then the bacteriophage is extracted through cell crushing, centrifugal precipitation, and filtration of the supernatant.
[0009] The double electrode system comprises an electrolytic cell, in which a direct current power supply, an anode, a cathode and a magnetic stirrer are arranged.
[0010] The bacterial electrolyte is arranged in the electrolytic cell, and the anode is a metal electrode and the cathode is a porous graphite electrode, so that more reaction sites are provided, a large amount of active substances are generated, and the electrochemical reaction rate is improved.
[0011] The anode is a noble metal electrode, including one of an IrO2 / RuO2 mixed coating electrode and a platinum electrode.
[0012] The cathode is a platinum-based electrode, a composite electrode or a graphite electrode, including one of a platinum electrode, a porous graphite electrode and a graphene-platinum plating electrode.
[0013] The effective electrode size is 5-100 cm. 2 The electrode spacing between the anode and the cathode is 0.5-20 cm.
[0014] In the scheme, under the action of a direct current field, the bacteria with electronegativity approach the surface of the metal anode, and under the oxidation of OH - plasma, the original bacteriophage in the bacteria is activated, the progeny virus is released, and the bacteria are lysed.
[0015] Preferably, the concentration of bacteria in the selected bacterial electrolyte is 10 2 -10 8 CFU / ml, so as to simulate the concentration of bacteria in the water environment.
[0016] Preferably, the selected bacterial electrolyte is a 0.01-2M Na2SO4 solution, so as to ensure the normal operation of the electrolysis system.
[0017] Preferably, the control voltage of the low-voltage electrolysis process is 0.5-10V, and the time is 1-200min, so as to ensure low energy consumption.
[0018] Preferably, the culture solution is a 0.1-8-fold concentrated culture medium, and the 1-fold culture solution is prepared by using 10g / L of tryptone, 4g / L of yeast extract, 10g / L of sodium chloride, 4g / L of glucose and 6g / L of calcium malate, and the pH is adjusted to 7.4 by using NaOH.
[0019] Preferably, the culture temperature is 25-40℃, and the time is 4-16h.
[0020] Preferably, the present invention uses the double-layer plate method to detect the number of phages generated after the original phage is activated.
[0021] Preferably, the cell disruption agent used is 0.1-5% (v / v) chloroform to disrupt the cell membrane structure and cause incompletely lysed bacteria to completely release the bacteriophage particles encapsulated inside.
[0022] Preferably, the centrifugation process for the centrifugation precipitation is performed using parameters of 8000-12000×g for 5-20 min to precipitate cells and cell debris.
[0023] Preferably, the supernatant is filtered using a 0.2 μm or 0.45 μm polyethersulfone filter membrane to remove residual cells and cell debris.
[0024] Preferably, the filtrate supernatant is serially diluted with 0.1M PBS for accurate bilayer plate counting. The experimental steps of the bilayer plate method are as follows: Mix 100 μL of serially diluted filtrate with 1 mL of host bacterial culture cultured for 5 h, and incubate at 37°C for 20 min. Then mix the mixture with 3 mL of upper plate, pour it onto the lower plate, seal after solidification, and incubate upside down at 37°C for 12-16 h. Select plates with 30-300 plaques for counting. The upper plate composition is 10 g / L tryptone, 1 g / L yeast extract, 8 g / L sodium chloride, adjust the pH to about 7.4, add 4.5 g / L agar, boil, and aliquot 3 mL per sample. The lower plate composition is 33 g / L nutrient agar, boil, and pour about 10 mL of each sterile medium.
[0025] The beneficial effects of this invention are:
[0026] The prophage is activated by generating highly active free radicals. The reaction process is simple, energy consumption is low, and reaction time is short.
[0027] This invention can simultaneously degrade organic matter in water while activating prophages for sterilization; the electro-activation of prophages is highly effective, with low technical cost and strong operability.
[0028] The method of this invention is environmentally friendly and avoids secondary pollution from chemical substances. Attached Figure Description
[0029] Figure 1 Phage plaques detected by the double-layer plate method after low-pressure electrolysis.
[0030] Figure 2 Images of bacteriophages observed by transmission electron microscopy after low-voltage electrolysis.
[0031] Figure 3 The number of phage plaques detected by the double-layer plate method after low-pressure electrolysis. Detailed Implementation
[0032] The application will be further described in detail below with reference to the accompanying drawings.
[0033] The application is applied to the activation of the original phage, and good results are achieved. The specific implementation is as follows:
[0034] Example 1
[0035] (1) Preparation of electrolyte: E. coli (ATCC 10798) containing original phage lambda and host E. coli (ATCC 23724) were selected, incubated with LB medium, eluted with 0.1M PBS to remove the remaining LB components in the solution, resuspended in 50mM Na2SO4 electrolyte, and diluted to an OD600 value of about 0.75.
[0036] (2) Reactor setup: 200ml of reaction solution was added, the anode was a Ti / RuIr electrode, the cathode was a carbon felt electrode, the effective electrode size was 2.5*2.0*0.1cm, and the electrode spacing was 2cm.
[0037] (3) Reaction conditions: the solution was exposed to a direct current voltage of 2V, and the exposure time was 0min, 15min, 30min, 45min, 60min, 75min, and 90min, respectively, and a certain amount of solution was collected at each stage to dilute in 1 times of culture medium and incubate for 5h.
[0038] (4) After incubation, 2% chloroform was added to the solution to break the cells, mixed well, and then stood for 5min, and then centrifuged at 8000xg for 5min. The supernatant was passed through a 0.22μm filter membrane, mixed with the host bacteria in the logarithmic phase, and then the number of progeny phages was detected by double-layer plate. After low-voltage electrolysis treatment, the number of progeny phages was as high as 1.24x10 5 PFU / ml.
[0039] Example 2
[0040] (1) Preparation of electrolyte: E. coli (ATCC 10798) containing original phage lambda and host E. coli (ATCC 23724) were selected, incubated with LB medium, eluted with 0.1M PBS to remove the remaining LB components in the solution, resuspended in 50mM Na2SO4 electrolyte, and diluted to an OD600 value of about 0.75.
[0041] (2) Reactor setup: 200ml of reaction solution was added, the anode was a Ti / RuIr electrode, the cathode was a carbon felt electrode, the effective electrode size was 2.5*2.0*0.1cm, and the electrode spacing was 2cm.
[0042] (3) Reaction condition: the solution was exposed to 2.5V direct current voltage, and the exposure time was 0min, 15min, 30min, 45min, 60min, 75min and 90min respectively, and a certain amount of solution was collected at each stage to dilute in 1 times of culture solution and cultivate for 5h.
[0043] (4) After incubation, 2% chloroform was added to the solution to break the cells, and after mixing, it was placed for 5min, and then centrifuged at 8000xg for 5min. The supernatant was filtered through a 0.22μm filter membrane, mixed with the host bacteria in the logarithmic phase, and then the number of progeny phages was detected by double-layer plate. After low-voltage electrolysis treatment, the number of progeny phages was as high as 1.72x10 5 PFU / ml.
[0044] Example 3:
[0045] (1) Configuration of electrolyte: E. coli (ATCC 10798) containing original phage lambda and host E. coli (ATCC 23724) were selected, incubated in LB medium, washed with 0.1M PBS to remove the remaining LB components in the solution, resuspended in 50mM Na2SO4 electrolyte, and diluted to about 0.75 OD600 value.
[0046] (2) Reactor setting: 200ml reaction solution was added, the anode was Ti / RuIr electrode, the cathode was carbon felt electrode, the effective electrode size was 2.5*2.0*0.1cm, and the electrode spacing was 2cm.
[0047] (3) Reaction condition: the solution was exposed to 3V direct current voltage, and the exposure time was 0min, 15min, 30min, 45min, 60min, 75min and 90min respectively, and a certain amount of solution was collected at each stage to dilute in 1 times of culture solution and cultivate for 5h.
[0048] (4) After incubation, 2% chloroform was added to the solution to break the cells, and after mixing, it was placed for 5min, and then centrifuged at 8000xg for 5min. The supernatant was filtered through a 0.22μm filter membrane, mixed with the host bacteria in the logarithmic phase, and then the number of progeny phages was detected by double-layer plate. After low-voltage electrolysis treatment, the number of progeny phages was as high as 1.18x10 5 PFU / ml.
[0049] As shown in Figure 1 , the phage plaques before and after the reaction detected by the double-layer plate method of the application show that a large number of original phages are activated by low-voltage electrolysis.
[0050] As shown in Figure 2 , the phage images observed by transmission electron microscope show the process of releasing original phages from the host and invading other hosts.
[0051] As Figure 3 The number of bacteriophage plaques detected by double-layer plate method shows that the voltage applied in the application can efficiently and quickly produce bacteriophages.
[0052] The above description is only the best mode of the application, but the protection scope of the application is not limited to this. Any person skilled in the art can easily think of substitution or change within the technical range disclosed by the application, which should be covered in the protection scope of the application.
Claims
1. A method for activating a pro-phage based on low voltage electrochemical technology, characterized in that, It comprises the following steps: The method comprises the following steps: adopting a double electrode system, low-voltage electrolysis of a bacterium electrolyte containing a bacteriophage, and after electrolysis, the reacted solution is cultured in a culture solution, and then the cells are broken, centrifugal precipitation is performed, the supernatant is filtered, and the bacteriophage is extracted.
2. A method of activating a pro-phage based on low-voltage electrochemical technology according to claim 1, characterized in that, The double electrode system comprises an electrolytic cell, in which a direct current power supply, an anode, a cathode and a magnetic stirrer are arranged; The bacterium electrolyte is arranged in the electrolytic cell, the anode is a metal electrode, and the cathode is a porous graphite electrode.
3. A method of activating a pro-phage based on low-voltage electrochemical technology according to claim 2, characterized in that, The anode is a noble metal electrode, which is one of an IrO2 / RuO2 mixed coating electrode and a platinum electrode; The cathode is a platinum-based electrode, a composite electrode or a graphite electrode, which is one of a platinum electrode, a porous graphite electrode and a graphene-platinum plating layer electrode. The effective electrode size is 5-100 cm 2 The electrode spacing of the anode and cathode is 0.5-20 cm.
4. The method of claim 1, wherein the method is based on low-voltage electrochemical activation of the original phage. The concentration of bacteria in the selected bacterial electrolyte was 10 2 -10 8 CFU / ml; The selected bacterium electrolyte is a 0.01-2M Na2SO4 solution; The control voltage of the low-voltage electrolysis process is 0.5-10V, and the time is 1-200min.
5. The method of claim 1, wherein the method is based on low voltage electrochemical technology to activate the original phage. The culture solution is a 0.1-8-fold concentrated culture medium, and the ratio of the 1-fold culture solution is as follows: 10g / L of tryptone, 4g / L of yeast extract, 10g / L of sodium chloride, 4g / L of glucose and 6g / L of calcium malate, and NaOH is used to adjust the pH to 7.4; The culture temperature is 25-40℃, and the time is 4-16h.
6. The method of claim 1, wherein the method is based on low voltage electrochemical technology to activate the original phage. The number of bacteriophages generated after the activation of the original bacteriophage is detected by a double-layer plate method.
7. A method of activating a pro-phage based on low-voltage electrochemical technology according to claim 6, characterized in that, The cell disrupter used for cell disruption is 0.1-5% (v / v) of chloroform; The selected parameters of the centrifugal process of the centrifugal precipitation are 8000-12000xg and 5-20min; The supernatant is filtered by using a 0.2μm or 0.45μm polyether sulfone filter membrane.
8. A method of activating a pro-phage based on low-voltage electrochemical technology according to claim 7, characterized in that, The filtered supernatant is gradiently diluted with 0.1M PBS to perform accurate double-layer plate counting; the experimental steps of the double-layer plate method are as follows: 100μL of the gradiently diluted filtrate is mixed with 1mL of a host bacterium solution cultured for 5h, and then the mixture is incubated at 37℃ for 20min. Then, the mixture is mixed with 3mL of an upper layer plate, poured on a lower layer plate, sealed after solidification, and incubated in a 37℃ incubator for 12-16h, and the plates with 30-300 bacteriophage plaques are selected for counting; the ratio of the upper layer plate is as follows: 10g / L of tryptone, 1g / L of yeast extract, 8g / L of sodium chloride, and the pH is adjusted to about 7.4, and then 4.5g / L of agar is added, and after boiling, 3mL of each sample is dispensed. The ratio of the lower layer plate is 33g / L of nutrient agar, and after boiling, about 10mL of each sterile culture medium is poured.