A method for controlling reverse osmosis membrane biofouling through synergistic control of quenching bacteria and bacteriophages.
By leveraging the synergistic effect of QQ bacteria and bacteriophages, a QQ-phage transport system was constructed, which solved the problem of reverse osmosis membrane biofouling, achieved efficient biofilm removal and membrane flux recovery, and extended the membrane's service life.
Patent Information
- Application Number
- CN202510141974.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-09
- Publication Date
- 2025-10-31
- Estimated Expiration
- 2045-02-09
AI Technical Summary
During operation, reverse osmosis membranes experience a decrease in membrane flux and a reduction in product water quality due to microbial biofouling. Traditional sterilization methods are ineffective in removing biofilm and can damage membrane materials, while replacing the membrane results in economic losses.
A QQ-phage transport system was constructed, which combines quorum quenching bacteria and bacteriophages. QQ bacteria degrade quorum sensing molecules to inhibit biofilm formation, and bacteriophages target and lyse the target biofouling bacteria to control reverse osmosis membrane biofouling.
It significantly improves biofilm removal efficiency, restores membrane flux, extends the service life of reverse osmosis membranes, and provides an efficient, green, and sustainable solution for biofouling control.
Smart Images
Figure CN119746636B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages. Background Technology
[0002] During reverse osmosis membrane operation, microorganisms accumulate on the membrane surface, causing biofouling, which affects system performance, leading to decreased membrane flux and reduced permeate quality. Biofilm formation is the main driving force of biofouling, a process regulated by quorum sensing (QS) systems. These systems regulate communication between microorganisms by secreting small, diffusible signaling molecules, thereby stimulating the production of extracellular polymers within the biofilm matrix. Common signaling molecules include acylhomoserine lactones (AHLs). Traditional sterilization methods often fail to eliminate biofilms; they damage membrane materials during cleaning; and disinfection byproducts are generated during the treatment process. The limitations of sterilizing agents mean that current applications often involve directly replacing the reverse osmosis membrane, resulting in significant economic losses.
[0003] Given the limitations of chemical disinfectants, phage strategies, which effectively target and lyse hosts and are membrane-friendly, have emerged as a novel bactericidal option. Phages are viruses that obligately infect prokaryotes. Virulent phages can self-replicate within infected host cells, assembling into new phage particles, then lysing the host cell and releasing new phages to infect neighboring cells. However, in real biofilms, hydraulic stress and the protective barrier of extracellular polymeric substances (EPS) limit phage infection. Quorum quenching (QQ) promotes biofilm dispersion by inhibiting the QS system, which promotes biofilm development. Therefore, a QQ-phage transport system composed of QQ bacteria and lysing phages was constructed, which is crucial for treating reverse osmosis membrane biofouling. Summary of the Invention
[0004] The purpose of this invention is to solve the problems existing in the prior art and to provide a method for the synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages.
[0005] The specific technical solution adopted in this invention is as follows:
[0006] This invention provides a method for the synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages, comprising the following steps:
[0007] S1. For the target biofouling bacteria to be controlled on the reverse osmosis membrane, the first screening condition is the ability to target and lyse the target biofouling bacteria, and the second screening condition is the ability to adsorb on quorum quenching bacteria with the ability to degrade quorum sensing molecules and form biofilms in a non-host form. Target phages that meet both screening conditions are screened from the phage library.
[0008] S2. The target phage is mixed and cultured with the quorum quenching bacteria, so that the target phage is adsorbed onto the quorum quenching bacteria in a non-host form to form a transport system.
[0009] S3. The transport system is applied to the surface of the reverse osmosis membrane containing the target biofouling bacteria. By quenching the bacteria to degrade quorum sensing molecules and inhibiting biofilm formation, the target phage targets and lyses the target biofouling bacteria, thereby controlling the biofouling on the reverse osmosis membrane.
[0010] As a preferred embodiment of the first aspect above, the target biofouling bacteria are core pollutants selected from biofilms attached to the surface of reverse osmosis membranes with membrane fouling using bacterial screening technology.
[0011] As a preferred embodiment of the first aspect above, the swarm quenching bacteria is Paenarthrobacter nicotinovorans, and the target biological contaminant bacteria for targeted lysis is Pseudomonas aeruginosa.
[0012] As a preferred embodiment of the first aspect above, the swarm quenching bacteria is nicotine-eating arthrobacterium ATCC 49919.
[0013] As a preferred embodiment of the first aspect above, the method for screening target phages from the phage library is as follows:
[0014] For different phages in the phage library, they were respectively mixed with the target biological pollutant bacteria in LB medium to isolate candidate phages that can infect and replicate using the target biological pollutant bacteria as host cells;
[0015] Each candidate phage was co-cultured with the population of quenched bacteria, and the presence of non-host free-riding adsorption behavior between each phage and the non-host strain was verified by transmission electron microscopy. If such behavior was found, the corresponding candidate phage was selected as the target phage.
[0016] As a preferred embodiment of the first aspect above, in step S3, after the reverse osmosis membrane contaminated by the target biological contaminant bacteria is removed from the reverse osmosis membrane assembly, the reverse osmosis membrane is treated offline using a solution containing the transport system to achieve biological control of the biological contamination on the surface of the reverse osmosis membrane.
[0017] As a preferred embodiment of the first aspect above, the offline treatment employs one or more of the following methods: spraying, rinsing, and soaking.
[0018] As a preferred embodiment of the first aspect above, in step S3, the solution containing the transport system is directly added to the feed water in the reverse osmosis membrane module to alleviate membrane fouling online without disassembling the reverse osmosis membrane that has been biofouled by the target biofouling bacteria.
[0019] As a preferred embodiment of the first aspect, when the transport system is applied to the surface of a reverse osmosis membrane containing the target biological contaminants for biological control, the pH value of the environment in which the transport system is located is controlled to be 6-8, so as to avoid the inhibitory effect of excessive acidity or alkalinity on the bacteriophages.
[0020] As a preferred embodiment of the first aspect above, the different phages in the phage library are obtained by isolating and purifying them from municipal wastewater.
[0021] As a preferred embodiment of the first aspect above, the municipal wastewater is the influent of the membrane module containing the reverse osmosis membrane to be controlled in the wastewater treatment facility.
[0022] Secondly, the present invention provides a transport system for controlling biofouling of reverse osmosis membranes, comprising a target bacteriophage adsorbed onto quorum quenching bacteria in a non-host form; the quorum quenching bacteria are nicotine-eating arthrobacteria; the target bacteriophage can target and lyse the target biofouling bacteria, and can also adsorb onto the quorum quenching bacteria in a non-host form. The target biofouling bacteria are preferably *Pseudomonas aeruginosa*.
[0023] Compared with the prior art, the present invention has the following advantages:
[0024] This invention constructs a QQ-phage transport system composed of QQ bacteria and lytic bacteriophages, which synergistically enhances the inhibition of biofilm formation and biofouling. In this QQ-phage transport system, the bacteriophages can target and lyse core polluting bacteria, while the QQ bacteria can degrade quorum-sensing molecules, weakening the bacteria's anti-phage defense system and promoting phage movement and infection within the biofilm. Therefore, this QQ-phage transport system can significantly improve biofilm removal efficiency, restore membrane flux, and extend the lifespan of reverse osmosis membranes. This invention can be applied in water treatment, seawater desalination, and industrial wastewater reuse, providing a highly efficient, green, and sustainable solution for biofouling control. Attached Figure Description
[0025] Figure 1 This is a diagram of the transport system construction under a transmission electron microscope.
[0026] Figure 2 To isolate the removal effect of QQ bacteria on quorum sensing signal molecules.
[0027] Figure 3 This study compares the biofilm removal effects of different treatment methods in biofilm plate culture experiments.
[0028] Figure 4 This is a comparison of reverse osmosis membrane flux recovery curves under different treatment methods in an offline processing experiment.
[0029] Figure 5 This study compares the flux recovery curves of reverse osmosis membranes under different treatment methods in an online processing experiment. Detailed Implementation
[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the present invention can be combined accordingly without mutual conflict.
[0031] In a preferred embodiment of the present invention, a method for controlling reverse osmosis membrane biofouling by synergistic use of quorum quenching (QQ) bacteria and bacteriophages is provided, the method comprising the following steps:
[0032] S1. For the target biofouling bacteria to be controlled on the reverse osmosis membrane, the first screening condition is the ability to target and lyse the target biofouling bacteria, and the second screening condition is the ability to adsorb on QQ bacteria with the ability to degrade quorum sensing molecules and form biofilms in a non-host form. Target phages that meet both screening conditions are screened from the phage library.
[0033] S2. The target bacteriophage is mixed and cultured with the QQ bacteria, so that the target bacteriophage is adsorbed onto the QQ bacteria in a non-host form to form a transport system.
[0034] S3. The transport system is applied to the surface of the reverse osmosis membrane containing the target biofouling bacteria. The QQ bacteria degrade quorum sensing molecules and inhibit biofilm formation, while the target bacteriophages target and lyse the target biofouling bacteria, thereby controlling the biofouling on the reverse osmosis membrane.
[0035] It should be noted that the target biofouling bacteria in this invention are biofilm-forming bacteria on the reverse osmosis membrane that require biocontrol. The specific species used depends on the core polluting bacteria on the reverse osmosis membrane and is not limited to any particular species. The methods described in S1-S3 above are general methods. Based on these methods, a transport system capable of preventing the formation of biofilms by different target biofouling bacteria can be constructed, and this transport system can be used for biocontrol of the biofilm. In this transport system, bacteriophages and QQ bacteria are used synergistically to enhance the inhibitory effect on the biofilm. Bacteriophages can carry QQ bacteria into the polluted biofilm, while QQ bacteria can promote bacteriophage motility and infection, and increase the number of bacteriophages.
[0036] Therefore, like the target contaminating bacteria, the target bacteriophage and QQ bacteria are not specific bacterial species; the specific species type needs to be screened against the target contaminating bacteria. Specifically, the target bacteriophage must meet the two screening criteria mentioned above. The first criterion is the ability to target and lyse the target contaminating bacteria. That is, the target bacteriophage should be able to infect the target contaminating bacteria and replicate and proliferate using the target contaminating bacteria as a host, thereby causing lysis of the target contaminating bacteria during replication and proliferation, and inhibiting the biofilm formed by the target contaminating bacteria. The second screening criterion is the ability to adsorb onto QQ bacteria, which possess the ability to degrade quorum sensing molecules and have weak biofilm formation capabilities, in a non-host form. It is important to note that in the second screening criterion, QQ bacteria merely act as an adsorption carrier for the bacteriophage's hitchhiking migration, but do not replicate and proliferate on QQ bacteria as a host to destroy them. In addition to being able to adsorb bacteriophages in a non-host form, the aforementioned QQ bacteria should also meet two other conditions. First, they should have a quenching effect on quorum sensing signal molecules (such as C4-HSL and 3-oxo-C12-HSL), inhibiting biofilm formation by degrading these molecules. Since the signal molecules are largely similar across different bacteria, they can be quenched by using appropriate QQ bacterial agents. Second, QQ bacteria themselves should have the ability to form biofilms on reverse osmosis membranes to adhere to the membrane surface and function continuously. However, the biofilm-forming ability of QQ bacteria on reverse osmosis membranes should be weak to avoid hindering the reverse osmosis process. As a preferred criterion, the biofilm-forming ability of QQ bacteria should be weaker than that of Bacillus licheniformis 143 (catalog number ATCC11859).
[0037] In this invention, for any type of biofilm contamination, the target biocontaminating bacteria, target bacteriophages, and non-host QQ bacteria can be screened in the following manner.
[0038] In embodiments of the present invention, target biofouling bacteria can be screened from biofilms attached to the surface of reverse osmosis membranes with membrane fouling using bacterial screening technology, and their biofilm-forming ability on the reverse osmosis membrane surface can be verified through recontamination experiments. However, biofilm-forming bacteria in biofilms are often a complex mixed bacterial community, and controlling each biofilm-forming bacterium is obviously impractical. Therefore, core polluting bacteria can be screened from the biofilms attached to the reverse osmosis membrane surface, that is, the dominant bacterial species in the biofilm can be used as target biofouling bacteria for biological control. Taking the common Pseudomonas aeruginosa biofilm attached to the reverse osmosis membrane surface as an example, the dominant bacterial species is Pseudomonas aeruginosa, and this core polluting bacterium can be used as the target biofouling bacteria for controlling the Pseudomonas aeruginosa biofilm. In addition, it should be noted that the actual extracted target biofouling bacteria may be one or more. If there are multiple types, a transport system can be constructed to control different target biofouling bacteria, or a transport system capable of simultaneously controlling different target biofouling bacteria can be constructed.
[0039] Furthermore, for any biofilm contamination, non-host QQ bacteria capable of quenching quorum sensing signal molecules and possessing weak biofilm formation ability can be identified through literature reports or actual experiments. Taking *Pseudomonas aeruginosa* biofilm as an example, the corresponding signal molecules are C4-HSL and 3-oxo-C12-HSL. QQ bacteria isolated using these as carbon sources are *Paenarthrobacter nicotinovorans*, which can target and quench the signal molecules of the target biocontaminating bacteria (i.e., *Pseudomonas aeruginosa*). In the embodiments of this invention, *Paenarthrobacter nicotinovorans* can be autonomously isolated or commercially available strains can be used, such as *Paenarthrobacter nicotinovorans* with catalog number ATCC 49919, which carries the AHL lactonease gene and can degrade quorum sensing molecules and inhibit *Pseudomonas aeruginosa* biofilm formation.
[0040] Furthermore, for any biofilm fouling on a reverse osmosis membrane module, the method for screening target phages from a phage library is as follows:
[0041] For different phages in the phage library, they were respectively mixed with the target biological pollutant bacteria in LB medium to isolate candidate phages that can infect and replicate using the target biological pollutant bacteria as host cells;
[0042] Each candidate phage was co-cultured with the QQ bacteria, and the presence of non-host free-riding adsorption behavior between each phage and the non-host strain was verified by transmission electron microscopy. If such behavior was found, the corresponding candidate phage was selected as the target phage.
[0043] It should be noted that the aforementioned phage library is composed of different types of phages. Theoretically, the more types of phages included, the better, as more target phages can be screened. The phage library in this invention can be composed of commercially available phages, or it can be obtained by screening and purifying samples such as water, soil, and sludge. However, an excessively large phage library increases the workload of screening and reduces efficiency.
[0044] In embodiments of the present invention, considering that water bodies contaminated by target biofouling bacteria contain a large number of target biofouling bacteria, as well as a series of bacteriophages capable of targeting and lysing the target biofouling bacteria, the water bodies contaminated by the target biofouling bacteria can be used as the object for extracting a phage library, so as to more efficiently find phages that meet the aforementioned two screening conditions. Since municipal sewage contains a large number of various biofouling bacteria and bacteriophages capable of targeting and lysing these biofouling bacteria, in embodiments of the present invention, municipal sewage from municipal sewage treatment facilities can be selected as the object for extracting a phage library to construct a diverse phage library, so as to screen for the desired target phages. Specifically, in the construction process, the influent of municipal sewage treatment facilities can be collected and the mixed phages contained therein can be extracted. Then, the extracted mixed phages can be separated and purified to obtain a series of pure phages to construct the aforementioned phage library, verifying whether each phage meets the aforementioned two screening conditions. This screening method can narrow down the scope of the phage library and quickly and accurately screen for the target phages.
[0045] It should be noted that bacteriophages are widely distributed in nature, therefore the target bacteriophage ultimately selected is not necessarily a single type. Due to the vast diversity of bacteriophages in nature, generally a series of target bacteriophages meeting the aforementioned two selection criteria can be screened. These target bacteriophages can be used individually or in combination as the final target bacteriophages for constructing the transport system. In this invention, the target bacteriophage only needs to meet the functional requirements of the response; there is no need to limit the type.
[0046] It should be noted that if there are multiple target biofouling bacteria causing reverse osmosis membrane fouling, when screening candidate phages by co-culturing them with the target biofouling bacteria in LB medium, only the core biofouling bacteria (the dominant species in the biofilm) need to be screened as candidate phages for infecting and replicating the host cell. This ensures that the final transport system can inhibit the core biofouling bacteria and thus destroy the biofilm.
[0047] In addition, when constructing the transport system, the mixing ratio of QQ bacteria and target phage can be optimized and adjusted according to the actual situation, preferably 1:1 to 1:10.
[0048] Furthermore, the method of adding the final transport system in this invention depends on the specific target of the disease. For severely fouled reverse osmosis membranes, offline treatment can be used, involving rinsing and soaking with a bacterial agent to reduce the adverse effects of hydraulic stress on phage infection. A preferred approach is to remove the reverse osmosis membrane contaminated by the target bacteria from the membrane module and then treat it offline using a solution containing the transport system through spraying, rinsing, or soaking to biologically control the biofouling on the membrane surface. For slightly fouled reverse osmosis membranes, online treatment can be used to alleviate fouling and extend the biofilm's lifespan. A preferred approach is to directly add the solution containing the transport system to the feed water in the reverse osmosis membrane module to alleviate membrane fouling online without disassembling the membrane contaminated by the target bacteria. Additionally, regardless of whether offline or online treatment is used, the transport system can be periodically introduced onto the reverse osmosis membrane surface during the treatment process. It is recommended that the pH of the environment in which the transport system operates be controlled between 6 and 8 to avoid the inhibitory effect of excessive acidity or alkalinity on phages.
[0049] The following examples, using *Pseudomonas aeruginosa* biofilms as an example, demonstrate how to construct a transport system composed of bacteriophages and QQ bacteria to address this type of biofilm contamination. In the examples described below, the transport system consists of bacteriophages adsorbing onto non-host QQ bacteria in a non-host form; the QQ bacteria are *Arthrobacter nicotine phage*; and the bacteriophages are bacteriophages screened from wastewater capable of infecting *Pseudomonas aeruginosa*. However, it should be noted that this embodiment is merely an example, and the present invention is not limited to using the specific bacteriophages and QQ bacteria extracted in this embodiment.
[0050] Example
[0051] The first step is to screen for target biological contaminants and QQ bacteria.
[0052] (1) In this embodiment, the reverse osmosis membrane module requiring biofouling control is located in the desalination equipment of a seawater desalination plant. When collecting biofouling samples from the surface of the reverse osmosis membrane, the reverse osmosis membrane sheet was cut into 1 cm² pieces, placed in sterile phosphate-buffered saline (PBS), and sonicated for 5 minutes to release biofilm bacteria. The sonicated suspension was serially diluted and spread on LB agar medium, and incubated at 30°C for 48 hours. 16S rRNA sequencing identified Pseudomonas aeruginosa (PA) as the dominant bacterial species. Therefore, PA was selected as the target biofouling bacterium in this embodiment.
[0053] (2) Screening of quorum-quenching bacteria: In this embodiment, *Paenarthrobacter nicotinovorans* (PN) was selected as the QQ bacteria, with catalog number ATCC 49919. Transmission electron microscopy showed that PN has a flagellar structure, indicating its motility. LC-MS / MS confirmed that PN can degrade C4-HSL and 3-oxo-C12-HSL, with degradation rates of 78.3% and 92.1% respectively after 8 hours. Therefore, the selected QQ bacteria PN has the ability to degrade quorum-sensing molecules and can form a weak biofilm on the reverse osmosis membrane surface.
[0054] The second step is to screen for compatible phages and construct a delivery system.
[0055] (1) Isolation of Pseudomonas aeruginosa bacteriophages from wastewater. Wastewater samples from municipal wastewater treatment facilities were collected, and particles larger than 0.22 μm were removed by centrifugation and filtration. The phage extract in the filtrate was then further concentrated by polyethylene glycol 8000 (PEG 8000) precipitation and resuspended in SM buffer (50 mmol / L Tris-HCl [pH 7.5], 0.1 mol / L NaCl, 8 mmol / L MgSO4, 0.01% gelatin) to obtain the initial phage stock solution. To remove any contaminants, phages were further isolated from the initial phage stock solution. Each phage underwent three purification cycles, and the phage titer was determined per milliliter of PFU using triplicate double-layer plaques. The purified phages were added to a phage library for subsequent screening.
[0056] (2) Culturing the phage-contaminating bacterial mixture. For each purified phage in the phage library, using the target contaminating bacterium PA as the host, it was cultured in LB medium for 24 h to determine whether it could replicate and proliferate using Pseudomonas aeruginosa as the host. If so, the final cultured mixture was centrifuged at 10000g for 10 min at 4°C, and residual cells were removed through a 0.22μm filter. The resulting mixture was stored as candidate phages at 4°C for further analysis. In this embodiment, this step yielded multiple candidate phages.
[0057] (3) When the *Arthrobacter nicotine-eating* PN cultured in LB medium is in the stable growth phase, the suspension is diluted to OD 600 = 0.1. 1 mL of the *Arthrobacter nicotine-eating* suspension is added to 45 mL of phage stock solution for each candidate phage, and incubated separately in a shaker (30℃, 130 rpm) to promote adhesion between the candidate phages and *Arthrobacter nicotine-eating* cells. Then, the mixture of *Arthrobacter nicotine-eating* cells and candidate phages is collected and centrifuged at 4000g for 10 min at 4℃. Transmission electron microscopy is used to verify the non-host-based free-riding behavior between each candidate phage and *Arthrobacter nicotine-eating* PN. The specific verification method is as follows: 5 μL of each 10... 9 PFU / mL phage suspension was loaded onto a carbon-coated copper grid for 10 minutes and stained with 2% (w / v) phosphotungstic acid for 1 minute. The stained samples were washed twice with water and air-dried for at least 30 minutes. Subsequently, the samples were observed by transmission electron microscopy to verify the carrier behavior between each candidate phage and the non-host strain *Arthrobacter nicotine*.
[0058] (4) A transport system was established by adsorbing bacteriophages onto *Arthrobacter nicotine-eating bacteria* in a non-host form. The system was then negatively stained with 2% (w / v) phosphotungstic acid and verified using transmission electron microscopy. A migration assay was subsequently performed on a double-layer agar plate to verify the construction of the transport system. A bacterial lawn was formed using contaminated strains and treated with the following settings: PBS (control group), bacteriophages alone, QQ bacterial PN alone, and a PN-P transport system composed of *Arthrobacter nicotine-eating bacteria* and bacteriophages were added to soft agar double-layer plates containing PA and SW. At least three parallel experiments were performed for each group, and the infected area was measured after 6, 12, and 24 hours of incubation.
[0059] In this embodiment, a candidate bacteriophage, denoted as PPSW1, was selected after verification and screening. This phage exhibits both hitchhiking behavior with the non-host strain *Arthrobacter nicotine-eating* and the ability to use the pathogen *PA* as a host bacterium. The purified phage PPSW1 was stored in SM buffer at 4°C. The phage titer was quantified using the double-layer plaque assay and expressed as plaque-forming units (PFU).
[0060] The third step is the experiment on handling reverse osmosis membrane biofouling using a transport system.
[0061] (1) Biofilm plate culture experiment: Biofilms of the biocontaminating bacteria PA identified in the first step were established in 96-well plates. The biofilms were then treated with the different groups described above, resulting in four groups: PBS (control group), PPSW1 phage alone (phage group), PN (PN group), and a PN-P transport system composed of PN and PPSW1 phages (transport system group). Eight parallel cultures were set up for each biofilm. After 6, 24, and 48 hours of treatment, biomass, polysaccharides, and proteins were determined using the crystal violet method, phenol-sulfuric acid method, and Coomassie brilliant blue spectrophotometry, respectively. Simultaneously, the transcriptome and metabolome were also measured.
[0062] (2) Offline treatment experiment: PA was inoculated onto the surface of a polyamide reverse osmosis membrane and cultured at 37°C for 48 hours to form a mature biofilm. Offline treatment was performed on the four groups described above. Specifically, the contaminated reverse osmosis membrane was immersed in PBS, bacteriophage PPSW1, QQ bacteria *Arthrobacter nicotine* PN, and a PN-P transport system composed of *Arthrobacter nicotine* PN and bacteriophage PPSW1, respectively. After 1, 2, 3, and 4 hours, the membrane was reinserted into a laboratory-constructed reverse osmosis membrane device to measure permeability and desalination rate. During the measurement, a confocal laser scanning microscope (CLSM) was used to observe the biofilm structure and cell count for each treatment. A 488nm laser and a 560nm laser were used; live cells emitted green light under 488nm laser excitation, while dead bacteria emitted red light under 560nm laser excitation. The biofilm on the treated reverse osmosis membrane was stained with SYTO 9 and propidium iodide (PI). The structure of the biofilm was then detected using a Zeiss LSM 880 and observed using Zeiss ZEN 2012 software.
[0063] (3) Online treatment experiment: Biofouling was induced by adding PA to the feed water of the laboratory-constructed reverse osmosis membrane device, so that the PA bacteria concentration in the feed water reached 10. 6 CFU / mL. When the membrane flux decreased by 30%, it indicated severe biofouling of the membrane. At this point, the contaminated feed water was removed and online cleaning was initiated. During the online cleaning process, following the procedures of the four groups mentioned above, PBS, PPSW1 bacteriophage, QQ bacteria nicotine-eating arthropathogen PN, and a PN-P transport system composed of nicotine-eating arthropathogen PN and PPSW1 bacteriophage were introduced into the feed water. The water flux and desalination rate were measured hourly, and DNA was extracted for metagenomic analysis.
[0064] The relevant experimental results of this embodiment are shown below.
[0065] Figure 1The successful construction of the transport system was demonstrated under transmission electron microscopy, and the bacteriophage PPSW1 was successfully adsorbed onto QQ bacteria nicotine-phage arthropathobacterium PN.
[0066] Figure 2 This study demonstrated the quenching effect of *Bacteroides q. var. nicotinicus* PN on corresponding signal molecules. The degradation of key quorum sensing molecules C4-HSL and 3-oxo-C12-HSL was quantitatively analyzed by liquid chromatography-tandem mass spectrometry. The results showed that the concentration of C4-HSL decreased by 64.5% and 56.1% after 2 and 8 hours, respectively. For 3-oxo-C12-HSL, the degradation was more significant, with only 46.9% and 18.7% remaining after 2 and 8 hours, respectively.
[0067] Figure 3 The biomass at 6, 24, and 48 hours after different treatments in the above biofilm plate culture experiments is shown. In the early stage of biofilm formation (6 hours), the removal efficiencies of the phage group and the transport system group were similar, at 67.4% ± 18.1% and 66.6% ± 10.6%, respectively. In contrast, the removal efficiency of the PN group was lower, at 42.2% ± 7.1%. As the biofilm further formed (24 hours), the advantage of the transport system group became more pronounced, with a removal efficiency of 76.9% ± 4.8%, compared to 66.2% ± 8.3% for the phage group and only 28.9% ± 13.1% for the PN group. In the biofilm maturation stage (48 hours), the transport system group maintained a higher removal efficiency of 87.7% ± 6.7%, while the removal efficiency of the phage group was 72.0% ± 3.1%. As biofilms develop from the early stage to the mature stage, the removal efficiency of host systems carrying bacteriophages steadily improves, while the improvement is limited in groups using only bacteriophages, and the performance of PN groups alone remains weak.
[0068] Figure 4The comparison of reverse osmosis membrane flux recovery curves under different treatment methods in the above offline treatment experiments is presented. The membrane recovery effect of the transport system group gradually increased over time. In the 1-2 hour time period, the water flux recovery rate was 62.5%±5.6% and 69.3%±5.7%, which was lower than the 66.3%±6.7% and 75.1%±4.2% observed in the phage group. This is because the hydraulic disturbance was smaller during the offline treatment process, allowing the phage to contact and lyse the bacteria on the biofilm surface. Over time, the advantage of the transport system group became increasingly significant. At 3 hours and 4 hours, the water flux recovery rate of the transport system group reached 86.4%±3.1% and 94.5%±2.8%, respectively, exceeding the 78.3%±3.3% and 80.0%±3.3% of the phage group. In contrast, the PN group was almost ineffective against severe biofouling, with membrane flux decreasing from an initial 47.0% ± 3.0% to 39.4% ± 4.4% at 2 hours, and further to 29.6% ± 4.6% at 4 hours.
[0069] Figure 5 The comparison of reverse osmosis membrane flux recovery curves under different treatment methods in the above online treatment experiments is presented. Under severely fouled conditions, the effects of both the PN group and the phage group were limited—the PN group exacerbated the fouling, with water flux decreasing to 65.3% ± 2.5%; while the phage group only recovered a small amount of membrane flux, with water flux at 73.3% ± 2.1%. In contrast, the transport system group recovered membrane flux to 86.0% ± 2.3%.
[0070] The embodiments described above are merely some preferred implementations 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 synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages, characterized in that, Includes the following steps: S1. For the target biofouling bacteria to be controlled on the reverse osmosis membrane, the first screening condition is the ability to target and lyse the target biofouling bacteria, and the second screening condition is the ability to adsorb on quorum quenching bacteria with the ability to degrade quorum sensing molecules and form biofilms in a non-host form. Target phages that meet both screening conditions are screened from the phage library. S2. The target phage is mixed and cultured with the quorum quenching bacteria, so that the target phage is adsorbed onto the quorum quenching bacteria in a non-host form to form a transport system. S3. The transport system is applied to the surface of the reverse osmosis membrane containing the target biofouling bacteria. By quenching the bacteria to degrade quorum sensing molecules and inhibiting biofilm formation, the target phage targets and lyses the target biofouling bacteria, thereby controlling the biofouling on the reverse osmosis membrane.
2. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 1, characterized in that, The target biofouling bacteria are core pollutants selected from biofilms attached to the surface of reverse osmosis membranes with membrane fouling using bacterial screening technology.
3. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 1, characterized in that, The quenching bacteria mentioned are Paenarthrobacter nicotinovorans, and the target biological contaminant bacteria for targeted lysis is Pseudomonas aeruginosa.
4. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 3, characterized in that, The quenching bacteria were identified as nicotine-eating arthrobacterium ATCC 49919.
5. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 1, characterized in that, The method for screening target bacteriophages from the bacteriophage library is as follows: For different phages in the phage library, they were respectively mixed with the target biological pollutant bacteria in LB medium to isolate candidate phages that can infect and replicate using the target biological pollutant bacteria as host cells; Each candidate phage was co-cultured with the population of quenched bacteria, and the presence of non-host free-riding adsorption behavior between each phage and the non-host strain was verified by transmission electron microscopy. If such behavior was found, the corresponding candidate phage was selected as the target phage.
6. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 1, characterized in that, In step S3, after the reverse osmosis membrane contaminated by the target biological contaminant bacteria is removed from the reverse osmosis membrane assembly, the reverse osmosis membrane is treated offline by spraying, rinsing, or soaking using a solution containing the transport system, thereby controlling the biological contamination on the surface of the reverse osmosis membrane.
7. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 1, characterized in that, In step S3, the solution containing the transport system is directly added to the feed water in the reverse osmosis membrane module to alleviate membrane fouling online without disassembling the reverse osmosis membrane that has been biofouled by the target biological fouling bacteria.
8. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 1, characterized in that, When the transport system is applied to the surface of a reverse osmosis membrane containing target biological contaminants for biological control, the pH value of the environment in which the transport system is located is controlled at 6-8 to avoid the inhibitory effect of excessive acidity or alkalinity on bacteriophages.
9. The method for synergistic control of reverse osmosis membrane biofouling by swarm quenching bacteria and bacteriophages as described in claim 1, characterized in that, The different phages in the phage library were isolated and purified from municipal wastewater.
10. A transport system for controlling biofouling of reverse osmosis membranes, characterized in that, It consists of a target bacteriophage adsorbed onto a swarm quenching bacteria in a non-host form; the swarm quenching bacteria are nicotine-eating arthrobacteria; the target bacteriophage can target and lyse the target biological contaminant bacteria, and can also adsorb onto the swarm quenching bacteria in a non-host form.