Method for cooperatively controlling biological pollution of reverse osmosis membrane by group quenching bacteria and bacteriophage

Abstract

The invention discloses a method for cooperatively controlling biological pollution of a reverse osmosis membrane by group quenching bacteria and bacteriophages. According to the invention, a QQ-bacteriophage carrying system composed of quorum quenching (QQ) bacteria and lysing bacteriophages is constructed, and the bacteriophages and the QQ bacteria are utilized to synergistically improve the inhibition effect on biofilm formation and biological pollution. In the QQ-bacteriophage carrying system, the bacteriophage can perform targeted lysis on core contaminating bacteria, and the QQ bacteria can degrade quorum sensing molecules, weaken an anti-bacteriophage defense system of the bacteria and promote movement and infection of the bacteriophage in a biological membrane. Therefore, the QQ-bacteriophage carrying system can remarkably improve the biological membrane removal efficiency, recover the membrane flux and prolong the service life of the reverse osmosis membrane. The invention can provide an efficient, green and sustainable biological pollution control solution for the fields of water treatment, seawater desalination, industrial wastewater reuse and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for controlling biological contamination of a reverse osmosis membrane by synergistically using quorum quenching bacteria and bacteriophages. Background Art

[0002] During the operation of the reverse osmosis membrane, microorganisms accumulate on the membrane surface and cause biofouling, affecting system performance, resulting in decreased membrane flux and reduced water quality. The formation of biofilm is the main driving force of biofouling, and this process is regulated by the quorum sensing (QS) system. These systems regulate communication between microorganisms by secreting small diffusible signal molecules, thereby stimulating the production of extracellular polymers in the biofilm matrix. Common signal molecules include acyl homoserine lactones (AHLs). Traditional sterilization methods often fail to eliminate biofilms; damage to membrane materials during the cleaning process; and the treatment process is accompanied by the generation of disinfection by-products. The limitations of disinfectants mean that reverse osmosis membranes are usually replaced directly during current applications, resulting in huge economic losses.

[0003] In the context of the defects of chemical disinfectants, the phage strategy that can effectively target and lyse the host and is membrane-friendly has become a new bactericide option. Phage is a virus that specifically infects prokaryotes. Virulent phages can replicate themselves in the infected host cells and assemble into new phage particles, then lyse the host cells and release new phages to continue to infect neighboring cells. However, in actual biofilms, hydraulic stress and the protective barrier of biofilm extracellular polymers (EPS) limit phage infection. Quorum quenching (QQ) promotes biofilm dispersion by inhibiting the QS system that promotes biofilm development. Therefore, a QQ-phage carrying system consisting of QQ bacteria and lytic phages was constructed, which is very important for the treatment of reverse osmosis membrane biofouling. Summary of the invention

[0004] The purpose of the present invention is to solve the problems existing in the prior art and provide a method for controlling the biological fouling of reverse osmosis membrane by synergistically using quorum quenching bacteria and bacteriophages.

[0005] The specific technical solutions adopted by the present invention are as follows:

[0006] The present invention provides a method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophages, which comprises the following steps:

[0007] S1. For the target biological contaminants to be controlled on the reverse osmosis membrane, the first screening condition is the ability to target and lyse the target biological contaminants, and the second screening condition is the ability to adsorb in a non-host form on the quorum quenching bacteria with the ability to degrade quorum sensing molecules and form biofilms, and the target phages that meet both screening conditions are screened from the phage library;

[0008] S2, mixing and culturing the target phage with the quorum-quenching bacteria, so that the target phage is adsorbed on the quorum-quenching bacteria in a non-host form to form a carrying system;

[0009] S3. Applying the carrying system to the surface of the reverse osmosis membrane where the target biological contaminants exist, degrading the quorum sensing molecules and inhibiting the formation of biofilms through quorum quenching bacteria, and at the same time lysing the target biological contaminants through target bacteriophages, thereby achieving control of biological contamination on the reverse osmosis membrane.

[0010] As a preferred embodiment of the first aspect, the target biological contamination bacteria are core contamination bacteria screened out from the biofilm attached to the surface of the reverse osmosis membrane with membrane contamination by using a bacterial screening technology.

[0011] As a preferred embodiment of the first aspect, the quorum quenching bacteria is Paenarthrobacter nicotinovorans, and the target biological contamination bacteria for targeted lysis is Pseudomonas aeruginosa.

[0012] As a preferred embodiment of the first aspect, the quorum quenching bacteria is Bacteroides nicotinicola ATCC 49919.

[0013] As a preferred embodiment of the first aspect, the method for screening target phage from the phage library is:

[0014] For different phages in the phage library, they are mixed and cultured with the target biological contamination bacteria in LB culture medium to isolate candidate phages that can infect and replicate with the target biological contamination bacteria as host cells;

[0015] Each candidate phage is mixed with the group-quenched bacteria, and transmission electron microscopy is used to verify whether there is non-host hitchhiking adsorption behavior between each phage and the non-host strain. If so, the corresponding candidate phage is used as the target phage.

[0016] As a preferred embodiment of the first aspect, in S3, after the reverse osmosis membrane contaminated by the target biological contaminants is removed from the reverse osmosis membrane assembly, the reverse osmosis membrane is treated offline using a solution containing the carrying system to biologically control the biological contamination on the surface of the reverse osmosis membrane.

[0017] As a preferred embodiment of the first aspect, the off-line treatment adopts one or more methods of spraying, flushing and soaking.

[0018] As a preferred embodiment of the first aspect, in S3, the solution containing the carrying system is directly added to the influent water of the reverse osmosis membrane module, so as to alleviate the membrane contamination online without disassembling the reverse osmosis membrane contaminated by the target biological contaminants.

[0019] As a preferred embodiment of the first aspect, when the carrying system is applied to the surface of a reverse osmosis membrane where target biological contaminants exist for biological control, the pH value of the environment in which the carrying system is located is controlled to be 6 to 8 to avoid the inhibitory effect of excessive acidity or alkalinity on bacteriophages.

[0020] As a preference of the first aspect above, the different bacteriophages in the phage library are separated and purified from municipal sewage.

[0021] As a preferred embodiment of the first aspect, the municipal sewage is the influent water of a membrane module where a reverse osmosis membrane to be controlled is located in a sewage treatment facility.

[0022] In a second aspect, the present invention provides a transport system for controlling biological contamination of reverse osmosis membranes, which is composed of a target bacteriophage adsorbed on a population quenching bacteria in a non-host form; the population quenching bacteria is Bacteroides nicotinicus; the target bacteriophage can target and lyse the target biological contamination bacteria, and can be adsorbed on the population quenching bacteria in a non-host form. The target biological contamination bacteria is preferably Pseudomonas aeruginosa.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention constructs a QQ-phage carrying system composed of QQ bacteria and lytic phages, which utilizes phages and QQ bacteria to synergistically improve the inhibitory effect on biofilm formation and biological pollution. In the QQ-phage carrying system, phages can target and lyse core contaminating bacteria, while QQ bacteria can degrade quorum sensing molecules, weaken the anti-phage defense system of bacteria, and promote the movement and infection of phages in biofilms. Therefore, the QQ-phage carrying system can significantly improve the biofilm removal efficiency, restore membrane flux, and extend the service life of reverse osmosis membranes. The present invention can be used in the fields of water treatment, seawater desalination, and industrial wastewater reuse, providing an efficient, green and sustainable biological pollution control solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Diagram for the construction of the transport system for transmission electron microscopy.

[0026] Figure 2 To isolate the removal effect of QQ bacteria on quorum sensing signal molecules.

[0027] Figure 3 This is a comparison of the biofilm removal effects under different treatment methods in the biofilm well plate culture experiment.

[0028] Figure 4 Comparison of reverse osmosis membrane flux recovery curves under different treatment methods in offline treatment experiments.

[0029] Figure 5 Comparison of reverse osmosis membrane flux recovery curves under different treatment methods in online treatment experiments. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0031] In a preferred embodiment of the present invention, a method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching (QQ) bacteria and bacteriophages is provided, the method comprising the following steps:

[0032] S1. For the target biological contaminants to be controlled on the reverse osmosis membrane, the first screening condition is the ability to target and lyse the target biological contaminants, and the second screening condition is the ability to adsorb in a non-host form on QQ bacteria that have the ability to degrade quorum sensing molecules and form biofilms, and the target phages that meet both screening conditions are screened from the phage library;

[0033] S2, mixing and culturing the target bacteriophage with the QQ bacteria, so that the target bacteriophage is adsorbed on the QQ bacteria in a non-host form to form a carrying system;

[0034] S3. Apply the carrying system to the surface of the reverse osmosis membrane where the target biological contaminants exist, degrade the quorum sensing molecules and inhibit the formation of biofilm by QQ bacteria, and at the same time, target phages are used to target and lyse the target biological contaminants, thereby achieving control of biological contamination on the reverse osmosis membrane.

[0035] It should be noted that the target biological contamination bacteria in the present invention are biofilm-forming bacteria on the reverse osmosis membrane that need to be biologically controlled. The specific needs depend on the core contamination bacteria on the reverse osmosis membrane, and are not limited to specific bacterial species. The method described in S1 to S3 above is a general method. Based on this method, a carrying system that can prevent and control the biofilm formed by the target biological contamination bacteria can be constructed for different target biological contamination bacteria, and the carrying system can be used to perform biological control on the biofilm. In this carrying system, bacteriophages and QQ bacteria are used to synergistically improve the inhibitory effect on biofilms, wherein bacteriophages can carry QQ bacteria into the contaminated biofilm, and QQ bacteria can promote bacteriophage movement and infection and promote the increase in the number of bacteriophages.

[0036] Therefore, like the target biological contamination bacteria, the target phage and QQ bacteria are not a specified strain, and the specific strain type needs to be screened for the target biological contamination bacteria. Among them, the target phage needs to meet the above two screening conditions. The first screening condition is the ability to target the lysis of the target biological contamination bacteria, that is, the target phage should be able to infect the target biological contamination bacteria and replicate and proliferate with the target biological contamination bacteria as the host, thereby causing the lysis of the target biological contamination bacteria during the replication and growth process, and having a certain inhibitory effect on the biofilm produced by the target biological contamination bacteria. The second screening condition is the ability to adsorb in a non-host form on QQ bacteria with the ability to degrade quorum sensing molecules and weak biofilm formation. It should be noted that in the second screening condition, the QQ bacteria only serve as an adsorption carrier for the phage to migrate by hitchhiking behavior, but will not replicate and proliferate with the QQ bacteria as the host to destroy the QQ bacteria. In addition, in addition to being able to adsorb phages in a non-host form, the above-mentioned QQ bacteria should also meet two other conditions. One is that it should have a quenching effect on quorum sensing signal molecules (such as C4-HSL, 3-oxo-C12-HSL) and inhibit biofilm formation by degrading quorum sensing molecules. For different bacteria, the signal molecules are generally the same, so they can be quenched by matching suitable QQ bacterial agents. The second is that the QQ bacteria themselves should have the ability to form a biofilm on the reverse osmosis membrane so that they can adhere to the surface of the reverse osmosis membrane and continue to work. However, the ability of QQ bacteria to form a biofilm on the reverse osmosis membrane should be a weak film-forming ability to avoid the biofilm of QQ bacteria from hindering the reverse osmosis process. As a preferred standard, the film-forming ability of QQ bacteria should be weaker than that of Bacillus licheniformis 143 (catalog number ATCC11859).

[0037] In the present invention, for any biofilm contamination, the target biological contamination bacteria, target bacteriophages, and non-host QQ bacteria can be screened in the following manner.

[0038] In an embodiment of the present invention, the target biological contamination bacteria can be screened from the biofilm attached to the surface of the reverse osmosis membrane with membrane contamination by bacterial screening technology, and its biofilm forming ability on the surface of the reverse osmosis membrane can be verified by re-contamination experiments. However, the biofilm-forming bacteria in the biofilm are often a complex mixed bacterial community. It is obviously unrealistic to control each type of biofilm-forming bacteria. Therefore, the core contamination bacteria can be screened from the biofilm attached to the surface of the reverse osmosis membrane, that is, the dominant bacteria in the biofilm are used as the target biological contamination bacteria for biological control. Taking the common Pseudomonas aeruginosa biofilm attached to the surface of the reverse osmosis membrane as an example, the dominant bacteria is Pseudomonas aeruginosa, and this core contamination bacteria can be used as the target biological contamination bacteria for controlling the Pseudomonas aeruginosa biofilm. In addition, it should be noted that the target biological contamination bacteria actually extracted may be one or more. If there are multiple types, the carrying system can be constructed separately to control different target biological contamination bacteria, and the carrying system that can simultaneously control different target biological contamination bacteria can also be constructed.

[0039] In addition, for any biofilm pollution, non-host QQ bacteria that can quench quorum sensing signal molecules and have weak biofilm formation ability can be found through literature reports or actual experiments. Taking the Pseudomonas aeruginosa biofilm as an example, the corresponding signal molecules are C4-HSL and 3-oxo-C12-HSL, and the QQ bacteria separated by this as a carbon source are Paenarthrobacter nicotinovorans, which can target and quench the signal molecules of the target biological contamination bacteria (i.e., Pseudomonas aeruginosa). In an embodiment of the present invention, the nicotinovorans Bacteroides can be isolated autonomously, or a commercially available strain can be used, such as the nicotinovorans Bacteroides with a catalog number of ATCC 49919, which carries an AHL lactonase gene, can degrade quorum sensing molecules and inhibit the formation of Pseudomonas aeruginosa biofilms.

[0040] In addition, for any biofilm contamination on the reverse osmosis membrane module, the method for screening the target phage from the phage library is as follows:

[0041] For different phages in the phage library, they are mixed and cultured with the target biological contamination bacteria in LB culture medium to isolate candidate phages that can infect and replicate with the target biological contamination bacteria as host cells;

[0042] Each candidate phage was mixed with the QQ bacteria and tested by transmission electron microscopy to determine whether there was non-host hitchhiking adsorption between each phage and the non-host strain. If so, the corresponding candidate phage was used as the target phage.

[0043] It should be noted that the above-mentioned phage library is a library composed of different types of phages. In theory, the more types of phages it contains, the better, and the more target phages that can be screened. The phage library in the present invention can be composed of commercial phages, or can be obtained by screening and purification from samples such as water, soil, and sludge. However, an overly large phage library will increase the workload for screening and reduce the efficiency.

[0044] In an embodiment of the present invention, it is considered that the water body contaminated by the target biological contaminant contains a large number of target biological contaminants, and also contains a series of phages that can target and lyse the target biological contaminants. Therefore, the water body contaminated by the target biological contaminant can be used as the object of extracting the phage library, so as to more efficiently find phages that meet the above two screening conditions. Since municipal sewage contains a large number of various biological contaminants and phages that can target and lyse these biological contaminants, in an embodiment of the present invention, municipal sewage in a municipal sewage treatment facility can be selected as the object of extracting the phage library to construct a wide variety of phage libraries, so as to screen and obtain the desired target phages. In the specific construction, the water influent of the municipal sewage treatment facility can be collected and the mixed phages contained therein can be extracted, and then the extracted mixed phages can be separated and purified to obtain a series of pure phages to construct the above phage library, and verify whether each phage meets the above two screening conditions. This screening method can narrow the scope of the phage library and quickly and accurately screen the target phages.

[0045] It should be noted that bacteriophages are widely present in nature, so the target bacteriophages finally screened out are not necessarily just one. Since there are many types of bacteriophages in nature, generally speaking, a series of target bacteriophages that meet the above two screening conditions can be screened out in the end, and these target bacteriophages can be used alone or in combination as the target bacteriophages for the final construction of the carrying system. The target bacteriophages in the present invention only need to meet the corresponding functional conditions, and there is no need to limit the type.

[0046] It should be noted that if there are multiple target biological contaminants that cause reverse osmosis membrane contamination, then when the phages are mixed with the target biological contaminants in LB culture medium to screen candidate phages, it is only necessary to screen out the core biological contaminants (the dominant bacteria in the biofilm) as candidate phages for infection and replication and proliferation of host cells to ensure that the final carrying system can inhibit the core contaminants of the biofilm and thus destroy the biofilm.

[0047] In addition, when constructing the carrying system, the mixing ratio of QQ bacteria and target phage can be optimized and adjusted according to actual conditions, preferably 1:1 to 1:10.

[0048] In addition, in the present invention, the method of adding the finally constructed carrying system when applied needs to be determined according to the corresponding control object. On the severely contaminated reverse osmosis membrane, offline treatment can be used, and the microbial agent can be used for flushing and soaking to reduce the adverse effects of hydraulic stress on phage infection. The preferred specific method is: after the reverse osmosis membrane contaminated by the target biological contamination bacteria is disassembled from the reverse osmosis membrane assembly, the reverse osmosis membrane is treated offline by spraying, flushing, soaking or one or more methods using a solution containing the carrying system to control the biological contamination on the surface of the reverse osmosis membrane. On the slightly contaminated reverse osmosis membrane, online treatment can be used to alleviate the pollution and increase the service life of the biofilm. The preferred specific method is: the solution containing the carrying system is directly added to the influent water in the reverse osmosis membrane assembly, and the membrane pollution is alleviated online without disassembling the reverse osmosis membrane contaminated by the target biological contamination bacteria. In addition, whether it is offline treatment or online treatment, the carrying system can be regularly introduced to the surface of the reverse osmosis membrane during the treatment process, and it is recommended that the pH of the environment where the carrying system is located needs to be controlled to 6 to 8 to avoid the inhibitory effect of excessive acid or alkali on the phage.

[0049] Taking the Pseudomonas aeruginosa biofilm as an example, an example is given to demonstrate how to construct a transport system composed of bacteriophages and QQ bacteria for such biofilm pollution. In the following example, the transport system is composed of bacteriophages adsorbed on non-host QQ bacteria in a non-host form; the QQ bacteria are Bacteroides nicotinicus; and the bacteriophages are bacteriophages screened from wastewater that can infect Pseudomonas aeruginosa. However, it should be noted that this example is only an example, and the present invention is not limited to the use of bacteriophages and QQ bacteria specifically extracted in this example.

[0050] Example

[0051] The first step is to screen the target biological contaminants and QQ bacteria

[0052] (1) The reverse osmosis membrane assembly that needs to control biological contamination in this embodiment is located in the desalination equipment of a seawater desalination plant. When collecting biological contamination samples from the reverse osmosis membrane surface, the reverse osmosis membrane is cut into 1 cm 2 The small pieces were placed in sterile phosphate buffered saline (PBS) and ultrasonically treated for 5 minutes to release biofilm bacteria. The ultrasonically treated suspension was gradient diluted and applied to LB agar medium and cultured at 30°C for 48 hours. The dominant strain was identified as Pseudomonas aeruginosa (PA) by 16S rRNA sequencing. Therefore, PA was used as the target biological contamination bacteria in this embodiment.

[0053] (2) Screening of quorum quenching bacteria: In this example, Paenarthrobacternicotinovorans (PN) was selected as QQ bacteria, and its catalog number is ATCC 49919. Transmission electron microscopy showed that PN had a flagellar structure, indicating its motility. LC-MS / MS confirmed that PN could degrade C4-HSL and 3-oxo-C12-HSL, with degradation rates of 78.3% and 92.1% after 8 hours, respectively. Therefore, the selected QQ bacteria PN has the ability to degrade quorum sensing molecules and can form a weak biofilm on the surface of the reverse osmosis membrane.

[0054] The second step is to screen the aptamer phage and build the delivery system

[0055] (1) Isolation of Pseudomonas aeruginosa phage from wastewater. Wastewater samples were taken from municipal wastewater treatment facilities, and particles larger than 0.22 μm were removed by centrifugation and filtration; then, the phage extract in the filtrate was 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 an initial phage stock solution. To remove any contaminants, phages were further isolated from the initial phage stock solution, and each phage was subjected to three purification cycles, and the phage titer was determined by triplicate double-layer plaques in PFU per milliliter. The different phages isolated and purified were added to the phage library for subsequent screening.

[0056] (2) Cultivate the mixture of phages and contaminating bacteria. For each purified phage in the phage library, use the target biological contaminating bacteria PA as the host and culture it in LB medium for 24 hours to determine whether it can replicate and proliferate using Pseudomonas aeruginosa as the host. If possible, centrifuge the final cultured mixture at 10000g for 10 minutes at 4°C, remove residual cells through a 0.22μm filter, and store it at 4°C as a candidate phage for further analysis. In this embodiment, this step obtained a variety of candidate phages.

[0057] (3) When the Nicotinic alkaline Bacteroides PN cultured in LB medium is in the stable growth phase, dilute the suspension to OD600 = 0.1, add 1 mL of Nicotinic alkaline Bacteroides suspension to 45 mL of phage stock solution of each candidate phage, and incubate them separately in a shaker (30°C, 130 rpm) to promote the adhesion of candidate phages to Nicotinic alkaline Bacteroides cells. Then, collect the mixture of Nicotinic alkaline Bacteroides cells and candidate phages and centrifuge at 4000g for 10 minutes at 4°C. Use transmission electron microscopy to verify the non-host hitchhiking behavior between each candidate phage and Nicotinic alkaline Bacteroides PN. The specific verification method is as follows: 5 μL of each concentration of 10 9 The 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; then, the samples were observed by transmission electron microscopy to verify the transport behavior between each candidate phage and the non-host strain Bacteroides nicotinicus.

[0058] (4) The phage is adsorbed on the nicotinic Bacteroides in a non-host form to form a transport system, and negative staining is performed with 2% (w / v) phosphotungstic acid, and then the transport system between the phage and the bacteria is verified by transmission electron microscopy imaging. Subsequently, a migration test is performed on a double-layer plate to verify the construction of the transport system. The contaminated strain is used to form a bacterial lawn, and the following settings are used for treatment: PBS (control group), phage alone, QQ bacteria PN alone, and PN-P transport system composed of nicotinic Bacteroides and phage are added to the soft agar double-layer plate containing PA and SW. At least 3 parallel tests were performed for each group, and the infection area was measured after 6 hours, 12 hours, and 24 hours of incubation.

[0059] In this example, a candidate phage that can hitchhike with a non-host strain of Bacteroides nicotinicus and use the pathogen PA as a host strain was screened and verified, and was recorded as phage PPSW1. The purified phage PPSW1 was stored in SM buffer at 4°C. The double plaque method was used to quantify the phage titer and expressed in plaque forming units (PFU).

[0060] The third step is to carry out the experiment of treating the biological pollution of reverse osmosis membrane with the transport system.

[0061] (1) Biofilm plate culture experiment: The biofilm of the biological contaminant PA determined in the first step was established in a 96-well plate, and then treated with the above different groups. A total of four groups were set up, namely: adding PBS (recorded as the control group), adding bacteriophage PPSW1 alone (this treatment group was recorded as the phage group), adding QQ bacteria Nicotinic acid-loving Bacteroides PN alone (this treatment group was recorded as the PN group), adding the PN-P transport system composed of Nicotinic acid-loving Bacteroides PN and bacteriophage PPSW1 (this treatment group was recorded as the transport system group). Each biofilm was set up with 8 parallel cultures. After 6, 24, and 48 hours of treatment, the biomass, polysaccharides, and proteins were determined by crystal violet method, phenol-sulfuric acid method, and Coomassie brilliant blue spectrophotometry, respectively. At the same time, the transcriptome and metabolome were measured.

[0062] (2) Offline treatment experiment: PA was inoculated on the surface of polyamide reverse osmosis membrane and cultured at 37℃ for 48 hours to form mature biofilm. Offline treatment was carried out in the above four groups. Specifically, the contaminated reverse osmosis membrane was immersed in PBS, bacteriophage PPSW1, QQ bacteria Bacteroides nicotinicus PN, and PN-P carrying system composed of Bacteroides nicotinicus PN and bacteriophage PPSW1. After 1, 2, 3 and 4 hours, it was put back into the reverse osmosis membrane device constructed in the laboratory to measure the water permeability and desalination rate. During the measurement, confocal laser scanning microscopy (CLSM) was used to observe the biofilm structure and cell count of each treatment. The 488nm laser line and 560nm laser line were used. The living cells emitted green light under the excitation of the 488nm laser line, and the dead bacteria emitted red light under the excitation of the 560nm laser line. 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 by Zeiss ZEN 2012 software.

[0063] (3) Online treatment experiment: PA was added to the influent of the laboratory-built reverse osmosis membrane device to induce biofouling, and the PA bacterial concentration in the influent reached 10 6 CFU / mL. When the membrane flux dropped by 30%, it indicated that the membrane had been seriously bio-fouled. At this time, the contaminated influent was removed and online cleaning began. During the online cleaning process, PBS, bacteriophage PPSW1, QQ bacteria Nicotinic acid-loving Bacteroides PN, and the PN-P carrying system composed of Nicotinic acid-loving Bacteroides PN and bacteriophage PPSW1 were introduced into the influent according to the practices of the above four groups. The water flux and desalination rate were measured once an hour, and DNA was extracted for metagenomic determination.

[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 on the QQ bacteria Bacteroides nicotinicus PN.

[0066] Figure 2 The quenching effect of QQ bacteria Bacteroides nicotinicus PN on the corresponding signal molecules was demonstrated. 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 hours and 8 hours, respectively. For 3-oxo-C12-HSL, the degradation was more significant, with only 46.9% and 18.7% remaining after 2 hours and 8 hours, respectively.

[0067] Figure 3 The biomass after 6, 24, and 48 hours under different treatments in the above biofilm well plate culture experiment is shown. In the early stage of biofilm formation (6 hours), the removal efficiencies of the phage group and the carrier 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%. With the further formation of the biofilm (24 hours), the advantage of the carrier system group became more obvious, with a removal efficiency of 76.9% ± 4.8%, while the phage group was 66.2% ± 8.3%, and the PN group was only 28.9% ± 13.1%. In the mature stage of the biofilm (48 hours), the carrier system group maintained a high removal efficiency of 87.7% ± 6.7%, while the removal efficiency of the phage group was 72.0% ± 3.1%. As the biofilm progressed from the early stage to the mature stage, the removal efficiency of the host system carrying phages steadily increased, while the group using phages alone showed limited improvement, and the performance of the PN alone group was consistently weak.

[0068] Figure 4The comparison of the reverse osmosis membrane flux recovery curves under different treatment methods in the above-mentioned offline treatment experiment is shown. The membrane recovery effect of the transport system group gradually increased over time. In the 1-2 hour 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 due to the smaller hydraulic disturbance during the offline treatment process, which enables the phage to contact and lyse the bacteria on the surface of the biofilm. Over time, the advantages of the transport system group gradually became significant. At 3 hours and 4 hours, the water flux recovery rates 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 the membrane flux decreasing from the initial 47.0% ± 3.0% to 39.4% ± 4.4% at 2 h and to 29.6% ± 4.6% at 4 h.

[0069] Figure 5 The comparison of the reverse osmosis membrane flux recovery curves under different treatment methods in the above online treatment experiment is shown. Under severe pollution conditions, the effects of the PN group and the phage group were limited - the PN group aggravated the pollution, and the water flux dropped to 65.3% ± 2.5%; while the phage group only recovered a small amount of membrane flux, with a water flux of 73.3% ± 2.1%. In contrast, the transport system group restored the membrane flux to 86.0% ± 2.3%.

[0070] The above-described embodiments are only some preferred implementations of the present invention, but are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for controlling reverse osmosis membrane biofouling by synergistic use of quorum quenching bacteria and bacteriophages, characterized in that: The following steps are involved: S1. For the target biological contaminants to be controlled on the reverse osmosis membrane, the first screening condition is the ability to target and lyse the target biological contaminants, and the second screening condition is the ability to adsorb in a non-host form on the quorum quenching bacteria with the ability to degrade quorum sensing molecules and form biofilms, and the target phages that meet both screening conditions are screened from the phage library; S2, mixing and culturing the target phage with the quorum-quenching bacteria, so that the target phage is adsorbed on the quorum-quenching bacteria in a non-host form to form a carrying system; S3. Applying the carrying system to the surface of the reverse osmosis membrane where the target biological contaminants exist, degrading the quorum sensing molecules and inhibiting the formation of biofilms through quorum quenching bacteria, and at the same time lysing the target biological contaminants through target bacteriophages, thereby achieving control of biological contamination on the reverse osmosis membrane.

2. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 1, characterized in that: The target biological contamination bacteria are core contamination bacteria screened out from the biofilm attached to the surface of the reverse osmosis membrane with membrane contamination by using a bacterial screening technology.

3. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 1, characterized in that: The quorum quenching bacteria are Paenarthrobacter nicotinovorans, and the target biological contamination bacteria for targeted lysis are Pseudomonas aeruginosa.

4. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 3, characterized in that: The quorum quenching bacteria is Bacteroides nicotinicus ATCC 49919.

5. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 1, characterized in that: The method for screening target phage from the phage library is: For different phages in the phage library, they are mixed and cultured with the target biological contamination bacteria in LB culture medium to isolate candidate phages that can infect and replicate with the target biological contamination bacteria as host cells; Each candidate phage is mixed with the group-quenched bacteria, and transmission electron microscopy is used to verify whether there is non-host hitchhiking adsorption behavior between each phage and the non-host strain. If so, the corresponding candidate phage is used as the target phage.

6. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 1, characterized in that: In 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 at least one of spraying, flushing, and soaking using a solution containing the carrying system to perform biological control on the biological contamination on the surface of the reverse osmosis membrane.

7. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 1, characterized in that: In S3, the solution containing the carrying system is directly added to the influent water in the reverse osmosis membrane module, so as to alleviate the membrane pollution online without disassembling the reverse osmosis membrane polluted by the target biological pollutants.

8. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 1, characterized in that: When the carrying system is applied to the reverse osmosis membrane surface where the target biological contaminants exist for biological control, the pH value of the environment in which the carrying system is located is controlled to be 6-8 to avoid the inhibitory effect of excessive acid or alkalinity on bacteriophages.

9. The method for controlling reverse osmosis membrane biofouling by synergistically using quorum quenching bacteria and bacteriophage as claimed in claim 1, characterized in that: The different phages in the phage library are isolated and purified from municipal sewage.

10. A transport system for controlling biological fouling of reverse osmosis membranes, characterized in that: The target bacteriophage is adsorbed on the group-quenching bacteria in a non-host form; the group-quenching bacteria are nicotinic alkaloids Bacteroides; the target bacteriophage can target and lyse the target biological contaminant bacteria, and can also be adsorbed on the group-quenching bacteria in a non-host form.

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