Population quenching bacteria, population quenching particles as well as preparation method and application of population quenching particles

Abstract

The invention particularly discloses population sensing quenching bacteria, population sensing quenching particles and a preparation method and application of the population sensing quenching bacteria and the population sensing quenching particles. The preparation method comprises the following steps: dissolving sodium alginate and gelatin in water, completely dissolving, uniformly mixing, sterilizing, and cooling to obtain a mixed solution; uniformly mixing the ochrobactrum pseudoochrobactrum bacterial liquid and the mixed liquid to obtain a first bacterial liquid mixed liquid; filling the first bacterial liquid mixed solution into an injector of a 3D biological printer, and printing to obtain an inner core; uniformly mixing the rhodococcus liquid and the mixed liquid to obtain a second bacterial liquid mixed liquid, then loading the second bacterial liquid mixed liquid into an injector of a 3D biological printer, constructing a biological shell on the surface of an inner core to obtain gel particle small balls A, then immersing the gel particle small balls A into a cross-linking agent I, taking out the gel particle small balls A after cross-linking and curing, and washing the gel particle small balls A for three times to obtain gel particle small balls B; filling the mixed solution into an injector of a 3D biological printer, then coating the surfaces of the gel particle small balls B with films to obtain gel particle small balls C, and then immersing the gel particle small balls C into a cross-linking agent II for cross-linking and curing to obtain the group sense quenching particles.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a swarm-sensing quenching sterilizer, a swarm-sensing quenching granule, its preparation method, and its application. Background Technology

[0002] The activated sludge process is a commonly used biological wastewater treatment system in wastewater treatment plants, used to treat domestic and industrial wastewater. However, its stable operation is often disrupted by filamentous bulking caused by the overgrowth of prokaryotic bacteria or eukaryotic fungi. Filamentous bulking leads to many problems, including poor sludge settling performance, deterioration of effluent quality, and in severe cases, even collapse of the entire wastewater treatment system, posing a significant threat to aquatic environmental safety.

[0003] Currently, methods for controlling activated sludge bulking mainly include adding chemical agents and adjusting process operating parameters. However, these traditional methods have certain limitations: adding oxidants (such as sodium hypochlorite, hydrogen peroxide, ozone, etc.) can effectively control sludge bulking, but due to the non-selectivity of oxidants, it can also damage the functional bacteria in wastewater treatment, even leading to sludge floc disintegration, effluent quality deterioration, and problems such as difficulty in accurately controlling the dosage and high cost. Adjusting process operating parameters (such as shortening sludge age, increasing influent load, increasing aeration, etc.) can fundamentally inhibit filamentous bulking, but changes in process conditions also affect effluent quality. Shortening sludge age inhibits the growth of nitrifying bacteria, increasing influent load affects the treatment effect of pollutants, and increasing aeration makes the sludge floc structure loose and increases operating costs. Therefore, there is an urgent need for a green, low-carbon, and effective method to control filamentous bulking and ensure the stable operation of activated sludge systems in wastewater treatment.

[0004] Quorum sensing (QS) is an intercellular communication mechanism in which microorganisms monitor their own population density and regulate their collective behavior by secreting and sensing specific signaling molecules. In recent years, researchers have focused on the relationship between quorum sensing-mediated morphological changes and filamentous bacterial expansion in filamentous bacteria. Currently, acylated homoserine lactones (AHLs) have been shown to play a crucial role in promoting filamentous bacterial proliferation. Quorum quenching (QQ) is a method to inhibit microbial functional behavior by interfering with or blocking quorum sensing signal transduction.

[0005] However, the primary method of suppressing quorum sensing of filamentous bacteria by adding exogenous quorum quenching inhibitors requires continuous external application of these inhibitors, which undoubtedly leads to significant operating costs. Furthermore, long-term application of quorum quenching inhibitors may alter the microbial community, affecting effluent quality or causing biotoxicity. Therefore, how to suppress filamentous bacterial bulking in a low-cost, efficient, and long-term stable manner, while ensuring the stability of the activated sludge microbial community, is a technical problem that those skilled in the art hope to solve. Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a swarm-sensing quenching sterilization method, a swarm-sensing quenching granule, a preparation method thereof, and its application. This invention can inhibit the bulking of filamentous bacteria in a low-cost, efficient, and long-term stable manner, thus ensuring the stability of the activated sludge microbial community.

[0007] The technical solution of this invention is implemented as follows: A swarm-sensitizing quenching agent, wherein the swarm-sensitizing quenching agent is *Pseudomonas pallida*, and the preservation number of *Pseudomonas pallida* is CICC 21953.

[0008] A method for preparing swarm-quenching particles specifically includes the following steps: (1) Dissolve sodium alginate and gelatin in water, mix them completely, and then sterilize and cool to obtain a mixture; (2) The bacterial solution of the swarm-sensing quenching as described in claim 1 and the mixture obtained in step (1) are mixed evenly at a volume ratio of 1:9 to obtain the first bacterial solution mixture, and then stored at 4-6℃ for later use. (3) The first bacterial solution mixture obtained in step (2) is loaded into the syringe of the 3D bioprinter and the kernel is printed. (4) Take the Rhodococcus bacteria solution and the mixture obtained in step (1) and mix them evenly at a volume ratio of 1:9 to obtain the second bacterial solution mixture. Then, put the second bacterial solution mixture into the syringe of the 3D bioprinter and build a biological shell on the core surface to obtain gel particle spheres A. (5) Immerse gel particle microspheres A in a calcium chloride solution with a mass-volume ratio of 2-4% and crosslink and cure at 25-35℃ for 6-12 hours. Then take them out and wash them with water to obtain gel particle microspheres B. (6) The mixture obtained in step (1) is loaded into the syringe of the 3D bioprinter, and then coated on the surface of the gel particle microsphere B to obtain gel particle microsphere C; (7) The gel particles C are immersed in a mixed solution of 0.5-1% genipin and 2-4% calcium chloride by mass volume, and then crosslinked and cured at 25-35℃ for 12-24h to obtain the swarm quenching particles.

[0009] Furthermore, in step (1), the molecular weight of the gelatin is 50-100kDa and the Bloom strength is 200-250g.

[0010] Further, in step (1), the mass-volume percentage of sodium alginate in the mixture is 3-4%, and the mass-volume percentage of gelatin is 6-10%.

[0011] Further, the specific steps of step (1) are as follows: dissolve sodium alginate and gelatin in water respectively, mix the two solutions after they are completely dissolved, heat and stir at 80-90℃ until they are evenly mixed, and then sterilize at 121℃ for 20-30 minutes using a high-pressure steam sterilizer.

[0012] Furthermore, the OD600 value of the swarm-quenching bacterial solution in step (2) is 1.0-1.2; and the OD600 value of the Rhodococcus bacterial solution in step (4) is 0.8-1.0.

[0013] Furthermore, the syringe used in steps (3), (4) and (6) has a capacity of 10 mL, a nozzle diameter of 410 μm, and printing parameters of 0.5 mm / s, 30℃, and 0-500 kPa.

[0014] A swarm quenching particle, wherein the swarm quenching particle is prepared by the aforementioned method for preparing a swarm quenching particle.

[0015] Application of a swarm-quenching granule method to inhibit filamentous sludge bulking.

[0016] Furthermore, before adding the quorum quenching granules to the activated sludge system, the quorum quenching granules are first placed in a 30-35℃ constant temperature water bath shaker and aerated for 6-8 hours using quorum quenching induction medium. Then, 0.1 mM EDTA is added to chelate calcium ions, and the culture is continued for 8-12 hours. Finally, the granules are washed three times with sterile phosphate buffer. The composition of the quorum quenching induction medium is as follows: 2 g / L NaCl; 2 g / L tryptone; 1 g / L yeast extract; 1 g / L glucose; 100 μL / L γ-caprolactone.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The *Pseudomonas aeruginosa* with accession number CICC 21953 of this invention ( Pseudochrobactrum sp. It can eliminate AI-2 signaling molecules and secrete AHL lactonease to degrade short-chain C4-HSL, C6-HSL and other AHL signaling molecules, thus acting as a quorum quencher to inhibit the growth and reproduction of filamentous bacteria.

[0018] 2. This invention utilizes 3D bioprinting technology to construct swarm-quenching particles. These particles have a three-layer structure from the inside out: a core, a shell, and an outer membrane. The core contains *Pseudomonas aeruginosa*, and the shell contains *Rhodococcus* with accession number CCTCC AB 2015073. Rhodococcus sp. The outer membrane has relatively low porosity, while the outer shell and inner core have relatively high porosity. The higher porosity of the outer shell and inner core improves the mass transfer efficiency of nutrients and oxygen, providing sufficient space for the growth, reproduction, and stable metabolism of *Pseudomonas aeruginosa* and *Rhodococcus*. The lower porosity of the outer membrane ensures efficient diffusion of small-molecule nutrients from the outside to the outer shell and inner core, enhancing the exchange of substances between quorum sensing quenching bacteria and external nutrients and oxygen, ensuring the activity of quorum sensing quenching bacteria. It also improves the mass transfer efficiency of quorum sensing signal molecules, ensuring that quorum sensing quenching bacteria can effectively receive and quench signal molecules beneficial to quorum sensing, inhibiting the growth of filamentous bacteria. Simultaneously, it prevents external proteases, other bacteria, and other large molecules from invading the outer shell and inner core and competing for ecological niches with quorum sensing quenching bacteria, and helps reduce bacterial shedding and leakage.

[0019] Furthermore, the structure of the quorum sensing quenching particles of this invention effectively creates a dissolved oxygen concentration gradient. Specifically, the Rhodococcus bacteria inside the outer shell, as aerobic bacteria, consume oxygen from the surrounding environment, causing the oxygen concentration to gradually decrease from the outer shell to the core. This creates a suitable anaerobic environment for the facultative anaerobic Pseudocanobacterium in the core, resulting in both Rhodococcus and Pseudocanobacterium maintaining good activity. Thus, the aerobic Rhodococcus bacteria in the outer shell can remove DSF signaling molecules and secrete AHL acylase to degrade long-chain AHL signaling molecules such as 3OC12-HSL and C14-HSL. Meanwhile, the facultative anaerobic Pseudocanobacterium in the core removes AI-2 signaling molecules and secretes AHL lactonease to degrade short-chain AHL signaling molecules such as C4-HSL and C6-HSL. This allows for broad-spectrum coverage of quorum sensing signaling molecules, collectively enhancing the quorum sensing quenching ability and effectively inhibiting the growth and proliferation of filamentous bacteria such as planktonic sclerotia and microfilamentous bacteria, thereby achieving effective control of filamentous sludge bulking.

[0020] 3. This invention significantly improves the mechanical strength and stability of quorum quenching granules by controlling the ratio of sodium alginate and gelatin, thereby extending their service life in activated sludge systems. Simultaneously, precise spatial control achieved through 3D bioprinting technology ensures the uniform distribution of Rhodococcus and Pseudomonas aeruginosa within the quorum quenching granules, allowing them to fully function in their respective niches and enhancing the overall quorum quenching capability.

[0021] 4. The swarm-quenching granules described in this invention can stably and effectively inhibit the bulking of filamentous bacteria for a long period of time. The inhibition effect can be guaranteed without continuous external addition. Furthermore, the swarm-quenching bacteria fixed in the swarm-quenching granules will not produce biotoxicity in the activated sludge system, thus not affecting the stability and activity of other microbial communities. This avoids the secondary pollution problems that may be caused by traditional chemical agents, conforms to the green and low-carbon environmental protection concept, and avoids the limitations of traditional methods caused by adding chemical agents or adjusting process parameters. It has broad application value. Attached Figure Description

[0022] Figure 1 - A schematic diagram of the structure of the swarm-sensing quenching particles described in this invention.

[0023] Figure 2 -Graph showing the changes in sludge settling performance between the control group and the Pseudomonas aeruginosa group in Example 1.

[0024] Figure 3 - Macroscopic photograph of the swarm-sensing quenching particles obtained in Example 2.

[0025] Figure 4 -Illustrations showing the effects of inhibiting sludge bulking on the swarm-quenching particles obtained in Example 2, the gel particles obtained in Comparative Example 1, the Pseudomonas aeruginosa swarm-quenching particles obtained in Comparative Example 5, and the Rhodococcus swarm-quenching particles obtained in Comparative Example 6.

[0026] Figure 5 - The changing trends of the concentrations of various signal molecules when the sludge bulking was inhibited by the sludge quenching particles obtained in Example 2, the gel particles obtained in Comparative Example 1, the Pseudomonas aeruginosa sludge quenching particles obtained in Comparative Example 5, and the Rhodococcus spp. quenching particles obtained in Comparative Example 6.

[0027] Figure 6 - The changing trends of SVI and signal molecule concentrations over time when the sludge bulking was inhibited by the sludge quenching particles obtained in Example 2, the gel particles obtained in Comparative Example 1, the Pseudomonas aeruginosa sludge quenching particles obtained in Comparative Example 5, and the Rhodococcus spp. quenching particles obtained in Comparative Example 6.

[0028] Figure 7 - Comparison of the mechanical strength of the swarm-quenching particles prepared in Examples 2-3 and Comparative Examples 2-4.

[0029] Figure 8 - Comparison of the stability performance of swarm-sensing quenching particles prepared in Examples 2-3 and Comparative Examples 2-4.

[0030] Among them: 1-Group-sensing quenching particles; 2-Outer membrane; 3-Shell; 4-Core; 5-Three-dimensional network structure; 6-Pseudomonas aeruginosa; 7-Rhodococcus. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0032] A schematic diagram of the structure of the swarm-sensing quenching particles 1 prepared by this invention is shown below. Figure 1 As shown in the figure, the core 4 and shell 3 of the quorum sensing quenching particles have a three-dimensional network structure 5. *Pseudomonas aeruginosa* and *Rhodococcus* are uniformly distributed in the core and shell, respectively. The core and shell have high porosity, which is beneficial for improving the mass transfer efficiency of nutrients and oxygen, and provides sufficient space for the growth, reproduction, and stable metabolism of *Pseudomonas aeruginosa* 6 and *Rhodococcus* 7. The outer membrane 2 has a multi-layered microporous structure with low porosity, allowing small molecule nutrients to diffuse efficiently through the outer membrane to the shell and core, enhancing the exchange of substances between the quorum sensing quenching particles and external nutrients and oxygen, ensuring the activity of the quorum sensing quenching particles. It also improves the mass transfer efficiency of quorum sensing signal molecules, ensuring that bacteria can effectively receive and quench signal molecules beneficial to quorum sensing, inhibiting the growth of filamentous bacteria. Simultaneously, it prevents external proteases, other bacteria, and other large molecules from invading the shell and core and competing for ecological niches with the quorum sensing quenching particles, and also helps reduce bacterial shedding and leakage. This invention designs a core-shell structure and incorporates two different quorum-sensing quenching bacteria, *Pseudomonas aeruginosa* and *Rhodococcus*. This design ensures that the aerobic *Rhodococcus* in the outer shell can remove DSF signaling molecules and secrete AHL acylases to degrade long-chain AHL signaling molecules (such as 3OC12-HSL and C14-HSL), while the facultative anaerobic *Pseudomonas aeruginosa* in the inner shell can remove AI-2 signaling molecules and secrete AHL lactoneases to degrade short-chain AHL signaling molecules (such as C4-HSL and C6-HSL). This broad-spectrum coverage of quorum-sensing signaling molecules enhances the quorum-sensing quenching ability. Furthermore, the synergy between the two quorum-sensing quenching bacteria creates a micro-aerobic-anaerobic environment within the core-shell structure of the sphere, which is more conducive to maintaining high activity levels for both bacteria. This effectively inhibits the growth and proliferation of filamentous bacteria such as planktonic *Sphaerophytes* and *Microfilamentous*, thereby achieving effective control of filamentous sludge bulking.

[0033] Example 1 Two identical SBR reactors were prepared, one as a control group and the other as an experimental group (Pseudomonas aeruginosa group). In the experimental group, Pseudomonas aeruginosa bacterial solution was added to the SBR reactor (OD600 of Pseudomonas aeruginosa bacterial solution was 1.2, and the addition ratio was 10 mL / L). Then, bulking was induced under low dissolved oxygen conditions, and the changes in sludge settling properties during the bulking process were monitored.

[0034] Changes in sludge settling properties, such as Figure 2 As shown in Figure 2, the sludge settling properties of the *Pseudomonas aeruginosa* group were significantly improved compared to the control group. When the SVI value of the highly expanded control group exceeded 800 mL / g, the SVI value of the *Pseudomonas aeruginosa* group remained below 200 mL / g, indicating that *Pseudomonas aeruginosa* possesses quorum quenching capabilities.

[0035] Example 2 A method for preparing swarm-quenching particles specifically includes the following steps: (1) Sodium alginate (SA) and gelatin (Gel) (molecular weight 80kDa, Bloom strength 200g) were dissolved separately in deionized water. After complete dissolution, the two solutions were mixed and heated and stirred at 80°C until homogeneous. Then, the mixture was sterilized in an autoclave at 121°C for 30 minutes and cooled to obtain a mixed solution. The volume mass percentage of sodium alginate in the mixed solution was 3% and the volume mass percentage of gelatin was 8%. (2) Mix 1 mL of Pseudomonas aeruginosa bacterial solution (OD600 value of 1.2) with 9 mL of the mixture prepared in step (1), stir and mix at 30 °C for 30 min to obtain 10 mL of bacterial solution mixture I, and store at 4 °C for 24 h. The OD600 value of the Pseudomonas aeruginosa bacterial solution is 1.2. (3) The bacterial solution mixture I is loaded into the syringe of the 3D bioprinter, and the bacterial solution mixture I is extruded layer by layer through the syringe to vertically stack and construct the core of the gel particle spheres. The 3D bioprinting syringe has a capacity of 10 mL and a nozzle diameter of 410 μm. Its printing parameters are: printing speed of 0.5 mm / s, printing configuration and size set as a sphere with a radius of 0.8 mm, printing bed temperature of 30 ℃, and extrusion pressure range of 300 kPa; (4) Take 1 mL of Rhodococcus bacteria solution (OD600 value is 0.8) and mix it with 9 mL of the mixture prepared in step (1). Stir and mix at 30 °C for 30 min to obtain 10 mL of bacterial solution mixture II. According to step (3), construct a biological shell on the core surface of the gel particle microspheres using a 3D bioprinter. The shell thickness is 0.5 mm. The OD600 value of the Rhodococcus bacteria solution is 0.8. (5) The gel particles printed in step (4) are immersed in crosslinking agent I and crosslinked at 30 °C for 6 h for further curing. Then the gel particles are taken out and washed with water three times to obtain swarm quenching gel particles with a core-shell structure and a diameter of 2.6 mm. The crosslinking agent I is a 4% w / v calcium chloride solution.

[0036] (6) According to step (3), the mixture from step (1) is loaded into the syringe of the 3D bioprinter, and a film is coated on the outer shell surface of the core-shell structured gel particle microsphere. Then, the gel particle microsphere is immersed in crosslinking agent II and crosslinked at 30 °C for 12 h for deep curing. Finally, swarm quenching particles with an outer membrane-shell-core three-layer structure are obtained with a diameter of 3 mm. The crosslinking agent II is a mixed solution of 0.5% w / v genipin and 4% w / v calcium chloride.

[0037] Example 3 This embodiment is the same as Embodiment 2, except that in this embodiment, the volume mass percentage of sodium alginate in the mixture is 3% and the volume mass percentage of gelatin is 6%.

[0038] Example 4 This embodiment is the same as Embodiment 2, except that in this embodiment, the volume mass percentage of sodium alginate in the mixture is 3% and the volume mass percentage of gelatin is 10%.

[0039] Comparative Example 1 This embodiment is the same as Embodiment 2, except that in this comparative example, Pseudomonas aeruginosa and Rhodococcus were not added, and gel particles were prepared.

[0040] Comparative Example 2 This embodiment is the same as embodiment 2, except that in this comparative example, gelatin was not used in step (1), and the resulting mixture contained only sodium alginate, and the volume mass percentage of sodium alginate was 2%.

[0041] Comparative Example 3 This embodiment is the same as embodiment 2, except that in this comparative example, gelatin was not used in step (1), and the resulting mixture contained only sodium alginate, and the volume mass percentage of sodium alginate was 3%.

[0042] Comparative Example 4 This embodiment is the same as embodiment 2, except that in this comparative example, gelatin was not used in step (1), and the resulting mixture contained only sodium alginate, and the volume mass percentage of sodium alginate was 4%.

[0043] Comparative Example 5 This embodiment is the same as embodiment 2, except that in this comparative example, no Rhodococcus bacterial solution was added in step (4), and only Pseudomonas spp. quenching particles were prepared, namely Pseudomonas spp. quenching particles.

[0044] Comparative Example 6 This embodiment is the same as embodiment 1, except that in this comparative example, no Pseudomonas aeruginosa bacterial solution was added in step (2), and only Rhodococcus swarm quenching particles were prepared, namely Rhodococcus swarm quenching particles.

[0045] 1. Macroscopic photographs of the swarm-quenching particles obtained in Example 2 are shown below. Figure 3 As shown in the figure, the swarm quenching particles are milky white and have a regular spherical structure. They are semi-transparent, with a smooth surface without depressions or tails. Their diameter is about 3 mm, and the mass of a single swarm quenching particle is about 18 mg after wetting.

[0046] 2. After retrieving activated sludge from the aeration tank of a wastewater treatment plant in Chongqing, it was first aerated for 3 days, and then acclimated in an SBR reactor using artificially prepared simulated wastewater. After 11 days of cultivation, the effluent quality stabilized and met the standards. Then, the swarm-quenching granules obtained in Example 2 (referred to as the experimental group), the *Pseudomonas aeruginosa* swarm-quenching granules obtained in Comparative Example 5, and the *Rhodococcus* swarm-quenching granules obtained in Comparative Example 6 were added to three identical SBR reactors at a ratio of 0.27 g / L for reactor experiments. Simultaneously, an equal amount of gel particles obtained in Comparative Example 1 was added to the same SBR reactor as a control experiment, referred to as the control group. Finally, sludge settling performance was analyzed after 1, 3, 5...29 days of SBR reactor operation. Ultrasonic-assisted liquid-liquid extraction was used, and LC-MS (ultra-high performance liquid chromatography-triple quadrupole mass spectrometry) was used to quantitatively analyze the changes in the concentration of each signal molecule over time during sludge bulking after 11, 15, 17, 19, 21, 23, 25, and 29 days of SBR reactor operation.

[0047] Before adding the swarm-quenching granules, *Pseudomonas aeruginosa* swarm-quenching granules, and *Rhodococcus spp.* swarm-quenching granules to the SBR reactor, they were first placed in a 30°C constant temperature water bath shaker and cultured for 6 hours with swarm-quenching induction medium (2 g / L NaCl; 2 g / L tryptone; 1 g / L yeast extract; 1 g / L glucose; 100 μL / L γ-caprolactone) to activate the activity of *Rhodococcus spp.* swarm-quenching bacteria. Then, 0.1 mM EDTA was added to chelate Ca. 2+ Temporarily increase the permeability of the outer membrane and continue culturing for 8 hours to allow γ-caprolactone to diffuse further into the nucleus, providing abundant nutrients for the nucleus pseudoaneurysm, activating the activity of the nucleus pseudoaneurysm and thus improving its quenching activity. Finally, wash three times with sterile phosphate buffer to remove impurities and EDTA from the culture medium.

[0048] The simulated wastewater composition is as follows: 500 mg CH3COONa, 50 mg starch, 50 mg peptone, 30.7 mg KH2PO4, 152.9 mg NH4Cl, 90 mg MgSO4•7H2O, and 14 mg CaCl2•H2O are added per liter of water, along with 0.3 mL of trace elements per liter. The formula for trace elements per liter is as follows: 1.5 g FeCl3•6H2O, 0.15 g H3BO3, 0.03 g CuSO4•5H2O, 0.18 g KI, 0.12 g MnCl2•4H2O, 0.06 g Na2MoO4•2H2O, 0.12 g ZnSO4•7H2O, 0.15 g CoCl2•6H2O, and 10 g EDTA. The influent COD is 450-550 mg / L, TN is 50-60 mg / L, TP is 6-7 mg / L, and pH is 7.5-8.0.

[0049] The effects of the sludge bulking inhibition on the sludge bulking of the sludge bulking quenching particles obtained in Example 2, the gel particles obtained in Comparative Example 1, the *Pseudomonas aeruginosa* sludge bulking quenching particles obtained in Comparative Example 5, and the *Rhodococcus* sludge bulking quenching particles obtained in Comparative Example 6 are shown in the figure. Figure 4 As shown; the trends in the concentration changes of each signal molecule are as follows: Figure 5 As shown, where Figure 5 (a) Corresponding to the control group, Figure 5 (b) Corresponding to quenching particles of Rhodococcus spp. Figure 5 (c) Corresponding to *Pseudomonas aeruginosa* quenching particles, Figure 5 (d) Corresponding experimental group; the trends of SVI and AHLs signal molecule concentrations over time are as follows: Figure 6 As shown.

[0050] Depend on Figure 4 It is evident that the SBR reactor operates aerobically for the first 12 days to stabilize the reactor sludge; then it operates anoxically to induce activated sludge bulking. The sludge settling performance of the experimental group is significantly better than that of the control group, and the clump-sensing quenching granules obtained in Example 2 have the best effect.

[0051] Depend on Figure 5It is evident that, compared to the control group, the concentrations of DSF and long-chain AHL signaling molecules were significantly reduced in the Rhodococcus group. During the same time period, the concentration of DSF decreased by 5-6 ng / gVSS, and the concentration of long-chain AHL signaling molecules decreased by 6-15 ng / gVSS, indicating that the Rhodococcus group effectively eliminated DSF and long-chain AHL signaling molecules. The concentration of short-chain AHL signaling molecules was significantly reduced in the Pseudomonas group, decreasing by 8-30 ng / gVSS during the same time period, indicating that Pseudomonas can effectively eliminate short-chain AHL signaling molecules. In the experimental group, the concentrations of DSF, long-chain AHL signaling molecules, and short-chain AHL signaling molecules were all significantly reduced. Furthermore, compared to the Rhodococcus and Pseudomonas groups, the concentrations of DSF, long-chain AHL signaling molecules, and short-chain AHL signaling molecules in the experimental group were even lower, indicating that Rhodococcus and Pseudomonas have a synergistic effect in the quenching granules prepared using both Rhodococcus and Pseudomonas, enhancing the removal of signaling molecules.

[0052] Depend on Figure 6 As can be seen, when the sludge in the control group exhibited high bulking, with an SVI value exceeding 1000 mL / g, the SVI value in the experimental group remained consistently below 100 mL / g. Furthermore, the concentration of AHLs signaling molecules in the experimental group was significantly lower than that in the control group, the Rhodococcus group, and the Pseudomonas pseudocankertica group. While the signaling molecule concentration in the control group reached above 70 ng / g VSS, the concentration in the experimental group never exceeded 18 ng / g VSS. Therefore, this demonstrates that the sludge bulking quenching granules of the present invention can maintain good settling properties and effectively inhibit filamentous bacteria growth and control sludge bulking.

[0053] 3. A mechanical strength testing system was established based on the principle of hydrostatic deformation. Ten gel particle spheres were randomly selected, and after removing surface free moisture with dust-free filter paper, they were placed on a horizontal glass slide platform. Vertical pressure was applied to the particles by stacking standard weights. During the observation process, the load was continuously increased until the first particle underwent structural failure. The critical failure load value was recorded. This parameter characterizes the ultimate bearing capacity of the gel particles against mechanical stress. The mechanical strength of the swarm-quenching particles prepared in Examples 2-4 and Comparative Examples 2-4 was measured, and the results are as follows: Figure 7 As shown in the figure: (1) When the mixture contains only sodium alginate (SA), the mechanical strength of the quenching particles increases with the increase of SA concentration. When the volume mass percentage of SA is 4%, the mixture is relatively viscous and can be used to prepare quenching particles, but the preparation process is difficult. When the volume mass percentage of SA is ≥5%, the mixture is very viscous and cannot be used to prepare quenching particles.

[0054] (2) When gelatin was added to the 3% sodium alginate, the mechanical strength of the quenching particles was further improved. At the same time, the mechanical strength increased with the increase of gel concentration, but the increase became slower and slower.

[0055] 4. Long-term stability assessment through simulation of actual operating environment: Gel particles were added to a stably operating SBR reactor. Ten gel particle pellets were periodically taken, rinsed three times with deionized water, and surface free water was removed using clean filter paper. Their mass was immediately measured using an analytical balance. The structural stability of the particles in the complex biochemical environment was quantitatively characterized by the mass loss rate. The stability of the quorum quenching particles prepared in Examples 2-4 and Comparative Examples 2-4 was measured, and the results are as follows: Figure 8 As shown in the figure: (1) When only SA component is present, the mass of quenching particles changes significantly over time. In particular, the quenching particles with 2% SA are severely depleted during operation, and their mass drops sharply in a short period of time. After 10 days, the quenching particles are completely consumed, indicating that the stability of quenching particles prepared by using pure sodium alginate is poor.

[0056] (2) The mass of the quenching particles prepared in Examples 2-4 decreased slowly over time and remained at a high level after 45 days of operation, indicating that the quenching particles prepared by mixing sodium alginate and gelatin have excellent stability.

[0057] Finally, it should be noted that the above embodiments of the present invention are merely illustrative examples and not intended to limit the implementation of the invention. Those skilled in the art can make other variations and modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method of swarm-induced quenching, characterized in that, The swarm-quenching agent is *Pseudomonas pallida*, and the preservation number of *Pseudomonas pallida* is CICC 21953.

2. A method for preparing swarm-quenching particles, characterized in that, Specifically, the following steps are included: (1) Dissolve sodium alginate and gelatin in water, mix them completely, and then sterilize and cool to obtain a mixture; (2) The bacterial solution of the swarm-sensing quenching as described in claim 1 and the mixture obtained in step (1) are mixed evenly at a volume ratio of 1:9 to obtain the first bacterial solution mixture, and then stored at 4-6℃ for later use. (3) The first bacterial solution mixture obtained in step (2) is loaded into the syringe of the 3D bioprinter and the kernel is printed. (4) Take the Rhodococcus bacteria solution and the mixture obtained in step (1) and mix them evenly at a volume ratio of 1:9 to obtain the second bacterial solution mixture. Then, put the second bacterial solution mixture into the syringe of the 3D bioprinter and build a biological shell on the core surface to obtain gel particle spheres A. (5) Immerse gel particle microspheres A in a calcium chloride solution with a mass-volume ratio of 2-4% and crosslink and cure at 25-35℃ for 6-12 hours. Then take them out and wash them with water to obtain gel particle microspheres B. (6) The mixture obtained in step (1) is loaded into the syringe of the 3D bioprinter, and then coated on the surface of the gel particle microsphere B to obtain gel particle microsphere C; (7) The gel particles C are immersed in a mixed solution of 0.5-1% genipin and 2-4% calcium chloride by mass volume, and then crosslinked and cured at 25-35℃ for 12-24h to obtain the swarm quenching particles.

3. The method for preparing swarm-quenching particles according to claim 2, characterized in that, In step (1), the molecular weight of the gelatin is 50-100kDa and the Bloom strength is 200-250g.

4. The method for preparing swarm-quenching particles according to claim 2 or 3, characterized in that, In step (1), the mass-volume percentage of sodium alginate in the mixture is 3-4%, and the mass-volume percentage of gelatin is 6-10%.

5. The method for preparing swarm-quenching particles according to claim 2, characterized in that, The specific steps of step (1) are as follows: dissolve sodium alginate and gelatin in water respectively. After they are completely dissolved, mix the two solutions and heat and stir at 80-90℃ until they are evenly mixed. Then sterilize them at 121℃ for 20-30 minutes using a high-pressure steam sterilizer.

6. The method for preparing swarm-quenching particles according to claim 2, characterized in that, The OD600 value of the swarm-quenching bacterial solution in step (2) is 1.0-1.2; the OD600 value of the Rhodococcus bacterial solution in step (4) is 0.8-1.

0.

7. The method for preparing swarm-quenching particles according to claim 2, characterized in that, The syringe used in steps (3), (4) and (6) has a capacity of 10 mL, a nozzle diameter of 410 μm, and printing parameters of 0.5 mm / s, 30℃, and 0-500 kPa.

8. A swarm-quenching particle, characterized in that, It is prepared using the method described in any one of claims 2-7 for preparing swarm-quenching particles.

9. The application of the swarm-quenching granules described in claim 8 for inhibiting filamentous sludge bulking.

10. The application of the swarm-quenching granules according to claim 9 in inhibiting filamentous sludge bulking, characterized in that, Before adding the quorum quenching granules to the activated sludge system, the quorum quenching granules are first placed in a 30-35℃ constant temperature water bath shaker and aerated for 6-8 hours with quorum quenching induction medium. Then, 0.1 mM EDTA is added to chelate calcium ions, and the culture is continued for 8-12 hours. Finally, the granules are washed three times with sterile phosphate buffer. The composition of the quorum quenching induction medium is as follows: 2 g / L NaCl; 2 g / L tryptone; 1 g / L yeast extract; 1 g / L glucose; 100 μL / L γ-caprolactone.

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