A method for controlling the growth of biofilms in high hydraulic flow environments using UV-enhanced bacteriophages.

Abstract

This invention discloses a method for controlling the growth of biofilms in high hydraulic flow environments using ultraviolet-enhanced carrier phages, belonging to the field of water and wastewater treatment. The method comprises the following steps: (1) phage pretreatment and concentration; (2) phage library construction and preservation; (3) ultraviolet light-induced enhancement; and (4) biofilm control. This method screens out carrier phages from wastewater treatment systems, which can invade biofilms and other biological aggregates along with carrier bacteria. Based on this, ultraviolet light-induced enhancement is used to increase the colonization rate of the phages and their carrier bacteria on the host bacterial biofilm, thereby enhancing the control of host bacterial biofilm growth under high hydraulic flow conditions. This helps alleviate problems such as biofilm fouling, pipe network damage, and pathogen transmission caused by biofilms in wastewater treatment and pipe networks.

Description

Technical Field

[0001] This invention relates to a method for controlling biofilm growth in high hydraulic flow environments by ultraviolet light-enhanced biofilm control using transportable bacteriophages, belonging to the field of biological water and wastewater treatment. Background Technology

[0002] Biofilms are organized microbial aggregates attached to the surface of a material and encapsulated by extracellular macromolecular polymers. They provide a stable internal environment for cellular life activities and are an important life form for microorganisms. However, biofilms can lead to problems such as pathogen proliferation and accelerated corrosion of the attachment interface. In the practical application of membrane biotechnology for wastewater treatment and remediation of polluted water bodies, the accumulation and adhesion of biofilms at the membrane-water interface causes membrane biofouling, which in turn affects system performance.

[0003] Recent studies have confirmed that bacteriophages, as bacterial viruses, can enter biofilms through various mechanisms, including fluid entrainment and adsorption, enzymatic hydrolysis of macromolecules, and bacterial infection and lysis, thereby regulating biofilm community assembly and function. Compared to traditional biofilm control methods, the application of bacteriophage control avoids the introduction of exogenous antibiotics and bacteriostatic agents, making it a promising in-situ biofilm control strategy. Given the ecological characteristics and potential of bacteriophages in water and wastewater treatment systems, optimizing reliable bacteriophage biofilm control technologies is of great significance.

[0004] However, practical applications of phage-controlled biofilm technology often involve complex hydraulic flow regimes. In some scenarios, high hydraulic flow can significantly reduce the residence time of phages in the system, thereby decreasing their effectiveness. For example, in high hydraulic flow environments with a residence time of less than one minute, the control effect of phage administration is significantly reduced. Therefore, how to construct reliable phage biofilm control technology under high hydraulic flow environments is a pressing technical problem that needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a novel method for inhibiting biofilm growth in high-hydraulic-flow environments using ultraviolet-enhanced bacteriophages, thus providing a bacteriophage control method for biofilm-related problems in wastewater biological treatment systems. This method mainly includes the following steps: (1) bacteriophage pretreatment and concentration; (2) bacteriophage library construction and preservation; (3) ultraviolet-induced enhancement; and (4) biofilm control. This method utilizes the carrier effect of non-host bacteria and bacteriophages to enable bacteriophages to achieve better motility and biofilm penetration in high-hydraulic-flow environments. Furthermore, ultraviolet enhancement promotes the symbiotic relationship between bacteriophages and host bacteria, enhancing the bacteriophage's control effect on biofilm growth.

[0006] The specific technical solution of this invention is as follows:

[0007] A method for controlling biofilm growth in high hydraulic flow environments using UV-enhanced biophage-based biofilm control includes the following steps:

[0008] S1: The activated sludge is dispersed under ultrasonic and stirring treatment, and then the sludge is separated into sludge and water. The supernatant is filtered to remove bacteria and the filtrate containing bacteriophage is extracted. The filtrate is further concentrated and enriched through a tangential flow system to obtain bacteriophage enriched solution.

[0009] S2: Mix and disperse the non-host carrier bacteria that can adsorb bacteriophage with the bacteriophage enrichment solution. After the non-host carrier bacteria adsorb the bacteriophage, extract the precipitate containing the bacteriophage and non-host carrier bacteria by centrifugation. Then, resuspend and disperse the precipitate and remove the non-host carrier bacteria by filtration to obtain the bacteriophage solution.

[0010] S3: The carrier phage solution is remixed with the non-host carrier bacteria and added to an aquatic environment system colonized with the host bacterial biofilm for cultivation. The system is kept in a high hydraulic flow environment so that the carrier phage migrates with the non-host carrier bacteria and colonizes the host bacterial biofilm until the biofilm grows to maturity. In the early stage of cultivation, ultraviolet light needs to be applied to the system to enhance the infection and colonization of the carrier phage and the host bacteria. At other times, no ultraviolet light is required for light-protected cultivation.

[0011] Preferably, the specific procedure of S1 is as follows: the activated sludge is subjected to alternating ultrasonic vibration and magnetic stirring under an ice bath to fully disperse the sludge, and then the sludge is separated into sludge and water by natural sedimentation. The supernatant is collected and the process is repeated multiple times to fully remove sludge particles and aggregates. The supernatant obtained from the last sludge-water separation is retained. The collected supernatant is filtered sequentially through 0.45μm and 0.22μm microporous membranes to remove sludge impurities and bacteria. After resuspending in PBS solution, it is concentrated by a tangential flow concentration system. Finally, it is further concentrated and enriched by centrifugal ultrafiltration to obtain a phage enrichment solution.

[0012] Preferably, the ultrasonic oscillation treatment and magnetic stirring are performed alternately for at least three rounds.

[0013] Preferably, the specific procedure of S2 is as follows: non-host vector bacteria capable of adsorbing bacteriophages are cultured to the exponential growth phase, and then the bacterial solution is mixed with the bacteriophage enrichment solution and ultrasonically dispersed evenly. The mixture is then placed in a refrigerated environment to allow the non-host vector bacteria to adsorb the bacteriophages. The adsorbed mixture is then separated into solid and liquid phases by stepwise centrifugation. After centrifugation, the supernatant is removed, and the solid precipitate is resuspended in PBS solution. The bacteriophages are then dispersed in the PBS solution by ultrasonication. Finally, the mixture is filtered through a 0.22 μm filter membrane to obtain a carrier bacteria-bacteriophage mixture with broadcastability.

[0014] Preferably, the non-host carrier bacteria is a flagellate bacterium capable of adsorbing bacteriophages onto flagella.

[0015] Preferably, the non-host carrier bacteria are prepared by diluting activated sludge with PBS solution, spreading it on LB agar plates, culturing it overnight, and then selecting the largest colonies on the plate and screening them under a scanning electron microscope for flagellated bacteria capable of adsorbing bacteriophages as the dominant non-host carrier bacteria.

[0016] Preferably, the high hydraulic flow environment is a water environment system with a hydraulic residence time of less than 1 minute.

[0017] Preferably, the initial stage of cultivation is the first 5 to 10 minutes after the start of cultivation.

[0018] Preferably, the ultraviolet light is achieved by irradiation with a UV lamp.

[0019] Preferably, the optimal method of ultraviolet irradiation is as follows: irradiating the host bacterial biofilm with ultraviolet light through a UV lamp with a wavelength of 313–365 nm and a power of 5–20 W, at a distance of 5–10 cm, with a light intensity of 2.0–5.0 mW / cm² at the irradiated location. 2 The irradiation time is 5 to 10 minutes.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This method isolates and screens carrier phages from activated sludge. Ultraviolet (UV) irradiation promotes the colonization of these phages and their carrier bacteria on the host bacterial biofilm, thereby enhancing the phages' ability to control biofilm growth in high-hydraulic-flow environments. Using this method, the phage content in the biofilm system significantly increases, inhibiting the growth and maturation of the host bacteria. UV irradiation strengthens this effect, reducing the growth rate of the host bacterial biofilm and influencing the composition and structure of the biofilm surface. Therefore, this method is significant for controlling biofilm growth in high-hydraulic-flow environments and can help alleviate problems such as biofilm fouling, pipeline damage, and pathogen transmission caused by biofilms in wastewater treatment and pipe networks. Attached Figure Description

[0022] Figure 1 Flowchart for controlling biofilm growth in high hydraulic flow environments with UV-enhanced bacteriophage-borne phages;

[0023] Figure 2 The image shown is an electron micrograph of the vector bacteria-bacteriophage finally constructed in the example.

[0024] Figure 3The following are CLSM images of the biofilm control effect of carrier phages with and without UV enhancement in the examples: (a) Blank without carrier phages, (b) Control Group using only carrier phages, and (c) CLSM images of biofilm control using UV-enhanced carrier phage experimental groups.

[0025] Figure 4 The example illustrates the effect of bacteriophages on the regulation of EPS in mature biofilms. Detailed Implementation

[0026] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0027] The inventive concept of this invention involves screening bacteriophages with carrier-like properties from activated sludge. These phages can acquire the ability to migrate directionally with non-host bacteria through adsorption. In high-hydraulic-flow environments (such as sewage pipe flushing and membrane bioreactor cleaning processes), they can more accurately reach and colonize the target bacterial biofilm, thus achieving biofilm control. Furthermore, ultraviolet (UV) light irradiation can enhance the effect of bacteriophages in controlling biofilm growth through multiple pathways. In traditional methods, UV light irradiation is typically used to control mature biofilms, physically disrupting the biofilm and often requiring continuous irradiation, which consumes a significant amount of energy. The ultraviolet light enhancement method in this invention differs from traditional methods. It only requires short-term irradiation of the initial host bacterial biofilm. On the one hand, by disrupting the surface structure of the biofilm, it makes it easier for carrier bacteria and bacteriophages to enter and obtain more favorable ecological niches. On the other hand, since the interaction between host bacteria and bacteriophages is affected by environmental factors and non-host organisms, under ultraviolet light stress, the antagonistic effect between bacteriophages and hosts is weakened, and a potential synergistic effect is established to counteract environmental pressure, thereby increasing the concentration of bacteriophages in the biofilm and effectively controlling the growth of environmental host bacterial biofilms.

[0028] This invention aims to enhance the biofilm control effect of carrier phages in high-hydraulic-flow environments. Carrier phages are isolated and screened from activated sludge, and colonized on biofilms via migration from non-host carrier bacteria. Ultraviolet (UV) irradiation is used to enhance the colonization rate and biofilm control effect of the phages. The method mainly includes the following steps: (1) phage pretreatment and concentration; (2) phage library construction and preservation; (3) UV irradiation-induced enhancement; and (4) biofilm control.

[0029] like Figure 1 As shown, the specific implementation of this method is as follows:

[0030] S1: The activated sludge is dispersed under ultrasonic and stirring treatment, and then the sludge is separated into sludge and water. The supernatant is filtered to remove bacteria and the filtrate containing bacteriophages is extracted. The filtrate is further concentrated and enriched through a tangential flow system to obtain a bacteriophage enriched solution.

[0031] As a preferred embodiment of the present invention, the specific method of step S1 is as follows: the activated sludge is subjected to alternating ultrasonic vibration and magnetic stirring under ice bath to fully disperse the sludge, and then the sludge is separated into mud and water by natural sedimentation. The supernatant is collected and the process is repeated multiple times to fully remove sludge particles and aggregates. The supernatant obtained from the last sludge-water separation is retained. The collected supernatant is filtered through 0.45μm and 0.22μm microporous membranes to remove sludge impurities and bacteria. After resuspending with PBS solution, it is concentrated by a tangential flow concentration system. Finally, it is further concentrated and enriched by centrifugal ultrafiltration to obtain a phage enrichment solution.

[0032] As a preferred embodiment of the present invention, the ultrasonic oscillation treatment and magnetic stirring are performed alternately for at least three rounds.

[0033] S2: Mix and disperse the non-host carrier bacteria that can adsorb bacteriophages with the bacteriophage enrichment solution. After the non-host carrier bacteria adsorb bacteriophages, extract the precipitate containing the bacteriophages and non-host carrier bacteria by centrifugation. Then, resuspend and disperse the precipitate and remove the non-host carrier bacteria by filtration to obtain the bacteriophage solution.

[0034] As a preferred embodiment of the present invention, the specific steps of S2 are as follows: Non-host vector bacteria capable of adsorbing bacteriophages are cultured to the exponential growth phase. Then, the bacterial solution is mixed with the bacteriophage enrichment solution and ultrasonically dispersed evenly. The mixture is then placed in a refrigerated environment to allow the non-host vector bacteria to adsorb the bacteriophages. The adsorbed mixture is then centrifuged stepwise to achieve solid-liquid separation (during this process, bacteriophages not adsorbed on the vector bacteria enter the supernatant, while those adsorbed on the vector bacteria enter the precipitate). After centrifugation, the supernatant is removed, and PBS solution is added to the solid precipitate for resuspension. The bacteriophages are then dispersed in the PBS solution by ultrasonic dispersion. Finally, the solution is filtered through a 0.22 μm filter membrane to remove the non-host vector bacteria, resulting in a bacteriophage-carrying solution.

[0035] It should be noted that the non-host carrier bacteria in this invention do not refer to a specific species. Their function is to act as carrier bacteria to enhance the movement range of bacteriophages. Therefore, any bacteria capable of adsorbing bacteriophages onto their surface for movement is acceptable. In a preferred embodiment of this invention, the aforementioned non-host carrier bacteria are flagellated bacteria capable of adsorbing bacteriophages onto their flagella, such as *Bacillus cereus*. Of course, the non-host carrier bacteria can be a single species or a mixture of species; there is no limitation in this regard.

[0036] As a preferred embodiment of the present invention, the aforementioned non-host carrier bacteria can be prepared by the following method: Activated sludge is diluted with PBS solution and spread onto LB agar plates for overnight incubation. Then, several colonies with the largest diameter on the plate are selected, and flagellated bacteria capable of adsorbing bacteriophages are screened under a scanning electron microscope as the dominant non-host carrier bacteria. Since this method selects several colonies with the largest diameter on the plate, it indicates that the bacteria in these colonies have strong motility and high colonization efficiency, and the flagellated bacteria screened from them can be used to adsorb bacteriophages. Generally, 3 to 5 colonies with the largest diameter on the plate can be selected, and the colonies belonging to flagellated bacteria are merged through microscopic identification as non-host carrier bacteria.

[0037] S3: The carrier phage solution is remixed with the non-host carrier bacteria and added to an aquatic environment system colonized with the host bacterial biofilm for cultivation. The system is kept in a high hydraulic flow environment so that the carrier phage migrates with the non-host carrier bacteria and colonizes the host bacterial biofilm until the biofilm grows to maturity. In the early stage of cultivation, ultraviolet light needs to be applied to the system to enhance the infection and colonization of the carrier phage and the host bacteria. At other times, no ultraviolet light is required for light-protected cultivation.

[0038] It should be noted that the specific form of the aquatic environment system colonized with host bacterial biofilm in this invention is not limited and can be determined according to the actual application scenario, such as sewage pipe networks or membrane bioreactors. In the following embodiments, a simulated aquatic environment system is used, in which the host bacteria need to first colonize to a specific interface, and then the migration and infection of bacteriophages carrying the bacteria are simulated under high hydraulic flow conditions.

[0039] It should be noted that the high hydraulic flow environment in this invention is defined by the hydraulic residence time (HRT), which is an aquatic environment system with a hydraulic residence time of less than 1 minute. For a flow chamber, a high hydraulic flow environment can be simulated by pumping PBS buffer into it using a peristaltic pump. By changing the constant flow rate, the hydraulic residence time in the flow chamber can be controlled to be less than 1 minute.

[0040] It should be noted that the initial stage of cultivation in this invention refers to the first 5 to 10 minutes of cultivation.

[0041] In a preferred embodiment of the present invention, the aforementioned ultraviolet irradiation is achieved through UV lamp irradiation. Experiments have determined the optimal irradiation method to be: applying ultraviolet light to the host bacterial biofilm using a UV lamp with a wavelength of 313–365 nm and a power of 5–20 W, at an irradiation distance of 5–10 cm, with a light intensity of 2.0–5.0 mW / cm² at the irradiation location. 2 The irradiation time is 5 to 10 minutes.

[0042] In subsequent embodiments of the present invention, after the biofilm has grown to a mature stage, its composition and structure can be characterized to evaluate the efficiency of phage control over biofilm growth. In these subsequent embodiments, the biofilm structure is visualized using the Nikon A1-RsiCLSM; simultaneously, EPS from the mature host bacterial biofilm is extracted using a thermal extraction method to evaluate the effect of UV irradiation on enhancing phage control over the composition and structure of the host bacterial biofilm, and to determine the optimal UV light intensity and irradiation time.

[0043] The implementation process and effects of the present invention will be described in detail below with specific examples of phage screening and ultraviolet light-enhanced biofilm control under laboratory conditions.

[0044] Example

[0045] In this embodiment, the method for controlling the growth of high-hydraulic-flow biofilms using ultraviolet-enhanced bacteriophages specifically includes the following steps:

[0046] Step 1: Sludge sampling and supernatant concentration

[0047] 1 L of activated sludge was pretreated in an ice bath, followed by alternating ultrasonic treatment and magnetic stirring for thorough dispersion (5 minutes of ultrasonic treatment in the ice bath at 30 W and 47 kHz, and 50 rpm of magnetic stirring). After dispersion, the sludge was allowed to settle naturally for sludge-water separation, and the supernatant was collected. This process was repeated three times to ensure the removal of sludge particles and aggregates as much as possible, retaining the supernatant from the last sludge-water separation. The collected supernatant was then filtered sequentially through 0.45 μm and 0.22 μm microporous membranes to remove sludge impurities and bacteria. The filtered phage solution was then made up to 1 L with PBS and further concentrated to 100 mL using a tangential flow concentration system. Finally, the phage solution was concentrated to 25-30 mL by centrifugation and ultrafiltration to obtain the phage enrichment solution.

[0048] Step 2: Phage library construction and preservation

[0049] The activated sludge obtained from the wastewater treatment system was diluted 1000 times with PBS solution and spread on LB agar medium. The 3-5 largest colonies on the plate after overnight incubation were selected as candidate colonies with strong motility and high colonization efficiency. Flagellated bacterial colonies were screened under a scanning electron microscope and selected. The flagellated bacterial colonies were mixed and used as the dominant non-host carrier bacteria.

[0050] After screening for highly motile and efficient dominant non-host vector bacteria on LB agar, the selected non-host vector bacteria were cultured to the exponential growth phase (OD600 of 0.3–0.5). 2 mL of the bacterial culture was mixed with 18 mL of phage enrichment solution concentrated from sludge and ultrasonically dispersed. The mixture was then placed in a 4°C refrigerator for 8 hours to allow the non-host vector bacteria to adsorb the phage. The adsorbed mixture was then centrifuged at 200×g, 400×g, 600×g, 800×g, and 1000×g for 5 min, 5 min, 5 min, 5 min, and 10 min, respectively. After centrifugation, the non-carrying phages that did not adsorb onto the carrier bacteria were in the supernatant, while the phages that adsorbed onto the carrier bacteria were in the centrifugation precipitate. At this point, the supernatant was removed with a pipette, and PBS solution was added to make up to 10 mL. The precipitate was gently shaken to resuspend it, and ultrasonic dispersion was used to detach the phages from the carrier bacteria and disperse them in the solution. After filtering through a 0.22 μm filter membrane, the solution was stored to remove the carrier bacteria, thus obtaining the carryable phage solution.

[0051] Step 3: Enhance phage colonization with ultraviolet light irradiation

[0052] A 0.45 μm PVDF hydrophobic filter membrane was placed on LB hemi-agar medium as the biofilm growth interface. 3 mL of host bacterial culture was added to the filter membrane, and the culture dish was incubated at 20°C for one week to allow the host bacterial biofilm to grow on the filter membrane. The filter membrane was then placed at the bottom of a flow chamber (length × width × height = 2 cm × 2 cm × 3 cm). The carrier phage solution preserved in step 2 was remixed with non-host vector bacteria (OD600 0.3–0.5) cultured to the exponential growth phase at a 1:1 volume ratio to form a carrier-carrying phage mixture. LB liquid medium was then added to bring the LB medium concentration to 0.1%, forming a simulated mixture. The mixture was then placed at 4°C for 8 hours for adsorption. A peristaltic pump was used to pump this mixture into the flow chamber to simulate phage carrier-carrying and biofilm growth under high hydraulic flow conditions. The peristaltic pump maintained a constant flow rate of 20 mL / min, corresponding to a hydraulic residence time of 0.6 min, thus creating the high hydraulic flow conditions required for the simulation. In the initial stage of the simulation, the flow chamber is irradiated with ultraviolet light using a 313nm wavelength, 5W UV lamp at a distance of 5cm, with a corresponding light intensity of 2.0mW / cm². 2 The irradiation time is 10 minutes. After 10 minutes of irradiation, the ultraviolet light should be stopped and the culture should be carried out in the dark.

[0053] The electron micrograph of the finally constructed vector bacteria-carrying phage in this embodiment is shown below. Figure 2 As shown.

[0054] In addition, the above experiments are referred to as the experimental group. To compare the effects of ultraviolet light and carrier phage on biofilm growth, this embodiment includes a blank group and a control group. The blank group exhibits normal biofilm growth without the addition of a carrier bacteria-phage mixture or ultraviolet light enhancement. The control group, compared to the experimental group, does not use ultraviolet light enhancement in the initial stage of the simulation; instead, it is cultured in the dark.

[0055] Step 4: Biofilm Control

[0056] For the three sets of experiments above, a high hydraulic flow environment was maintained in the flow chamber to allow the biofilm to grow and mature. After the experiments, the filter membrane in the flow chamber was removed for sampling. The biofilm structure was visualized using a laser confocal microscope, and the results are as follows. Figure 3 As shown. From Figure 3 It was found that under enhanced ultraviolet light irradiation, the colonization rate of carrier bacteria and disseminated bacteriophages on the biofilm significantly increased, and biofilm growth was significantly inhibited, demonstrating a significant control effect. EPS from mature host bacterial biofilms was extracted using a thermal extraction method. Protein content was quantitatively analyzed using the BCA method, and polysaccharide content was quantitatively analyzed using the phenol-sulfuric acid method. The final results are as follows: Figure 4 As shown, compared with the system without UV enhancement, UV enhancement significantly reduced the protein content in mature biofilm EPS. Observation by laser confocal microscopy revealed that the thickness of the mature biofilm was significantly reduced under UV enhancement.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for controlling the growth of biofilms in high hydraulic flow conditions using ultraviolet-enhanced transportable bacteriophages, characterized in that, Includes the following steps: S1: The activated sludge is dispersed under ultrasonic and stirring treatment, and then the sludge is separated into sludge and water. The supernatant is filtered to remove bacteria and the filtrate containing bacteriophage is extracted. The filtrate is further concentrated and enriched through a tangential flow system to obtain bacteriophage enriched solution. S2: Mix and disperse the non-host carrier bacteria that can adsorb bacteriophage with the bacteriophage enrichment solution. After the non-host carrier bacteria adsorb the bacteriophage, extract the precipitate containing the bacteriophage and non-host carrier bacteria by centrifugation. Then, resuspend and disperse the precipitate and remove the non-host carrier bacteria by filtration to obtain the bacteriophage solution. S3: The carrier phage solution is remixed with the non-host carrier bacteria and added to an aquatic environment system colonized with the host bacterial biofilm for cultivation. The system is kept in a high hydraulic flow environment so that the carrier phage migrates with the non-host carrier bacteria and colonizes the host bacterial biofilm until the biofilm grows to maturity. In the early stage of cultivation, ultraviolet light needs to be applied to the system to enhance the infection and colonization of the carrier phage and the host bacteria. At other times, no ultraviolet light is required for light-protected cultivation. The non-host carrier bacteria were prepared by diluting activated sludge with PBS solution, spreading it on LB agar plates, and culturing it overnight. Then, the largest colonies on the plates were selected, and flagellated bacteria capable of adsorbing bacteriophages were screened under a scanning electron microscope as the dominant non-host carrier bacteria.

2. The method for controlling the growth of high-hydraulic-flow biofilms using UV-enhanced carrier phages as described in claim 1, characterized in that, The specific procedure of S1 is as follows: The activated sludge is subjected to alternating ultrasonic vibration and magnetic stirring under an ice bath to fully disperse the sludge. After natural sedimentation to achieve sludge-water separation, the supernatant is collected. This process is repeated multiple times to fully remove sludge particles and aggregates, and the supernatant obtained from the last sludge-water separation is retained. The collected supernatant is filtered sequentially through 0.45μm and 0.22μm microporous membranes to remove sludge impurities and bacteria. After resuspending in PBS solution, it is concentrated through a tangential flow concentration system. Finally, it is further concentrated and enriched by centrifugal ultrafiltration to obtain a phage enrichment solution.

3. The method for controlling the growth of high-hydraulic-flow biofilms using UV-enhanced carrier phages as described in claim 1, characterized in that, The ultrasonic oscillation treatment and magnetic stirring are performed alternately for at least three rounds.

4. The method for controlling biofilm growth in a high-hydraulic-flow environment using UV-enhanced carrier phages as described in claim 1, characterized in that, The specific steps of S2 are as follows: non-host vector bacteria capable of adsorbing bacteriophages are cultured to the exponential growth phase. Then, the bacterial solution is mixed with the bacteriophage enrichment solution and ultrasonically dispersed evenly. The mixture is then placed in a refrigerated environment to allow the non-host vector bacteria to adsorb the bacteriophages. The adsorbed mixture is then centrifuged stepwise to achieve solid-liquid separation. After centrifugation, the supernatant is removed, and the solid precipitate is resuspended in PBS solution. The bacteriophages are then dispersed in the PBS solution by ultrasonication. Finally, the mixture is filtered through a 0.226 μm filter membrane to obtain a carrier bacteria-bacteriophage mixture with broadcastability.

5. The method for controlling the growth of high-hydraulic-flow biofilms using UV-enhanced carrier phages as described in claim 1, characterized in that, The non-host carrier bacteria are flagellated bacteria capable of adsorbing bacteriophages onto their flagella.

6. The method for controlling the growth of high-hydraulic-flow biofilms using UV-enhanced carrier phages as described in claim 1, characterized in that, The high hydraulic flow environment is a water environment system with a hydraulic residence time of less than 1 minute.

7. The method for controlling the growth of high-hydraulic-flow biofilms using UV-enhanced carrier phages as described in claim 1, characterized in that, The initial stage of cultivation refers to the first 5-10 minutes after the start of cultivation.

8. The method for controlling the growth of high-hydraulic-flow biofilms using UV-enhanced carrier phages as described in claim 1, characterized in that, The ultraviolet light is achieved through irradiation by UV lamps.

9. The method for controlling the growth of high-hydraulic-flow biofilms using UV-enhanced carrier phages as described in claim 1, characterized in that, The optimal method of ultraviolet irradiation is as follows: irradiate the host bacterial biofilm with ultraviolet light using a UV lamp with a wavelength of 313-365nm and a power of 5-20W, at a distance of 5-10cm, with a light intensity of 2.0-5.0mW / cm² at the irradiated location. 2 The irradiation time is 5 to 10 minutes.

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