Pseudomonas and its isolation method and application

Abstract

The application belongs to the field of membrane pollution control, and particularly relates to a kind of pseudomonas and its separation method and application. The application provides a kind of pseudomonas, which is identified as Pseudomonas knackmussii HITSZ-Q1, is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO:66162. The strain is with C6-HSL as only carbon source and nitrogen source, and its 16S rDNA sequence is shown as SEQ ID NO.1. The strain has significant degradation capacity to C6-HSL, C8-HSL and 3-oxo-C12-HSL and other signal molecules, and the quenching rate can be up to 98.9%. The strain can effectively reduce the concentration of signal molecules of Pseudomonas aeruginosa and inhibit the formation of biofilm when co-cultured with Pseudomonas aeruginosa. Further, the strain is prepared into a carrier in the form of immobilized small balls, and is added to a membrane bioreactor, and the operation cycle is extended from 6.5 days to 10.5 days, and the membrane pollution delay rate is up to 61.5%.

Description

Technical Field

[0001] This invention belongs to the field of membrane fouling control, specifically relating to a Pseudomonas bacillus, its isolation method, and its application. Background Technology

[0002] Membrane bioreactors (MBRs) are wastewater treatment technologies that combine membrane separation units with biological treatment units. They are widely used due to their advantages such as high effluent quality, compact structure, and small footprint. However, unavoidable membrane fouling in sludge environments and frequent cleaning accelerate membrane module aging and increase operating costs. The causes of membrane fouling are complex. Among them, biofouling (i.e., biofilm formation on the membrane module surface) caused by the attachment and growth of microorganisms on the membrane module surface and their secreted extracellular substances is considered the most difficult fouling component to remove. This behavior of microorganisms is regulated by quorum sensing (QS). Microorganisms regulate the expression of density-dependent genes by producing signaling molecules, thereby modulating bacterial population characteristics, including bioluminescence, plasmid conjugation, and biofilm formation. Therefore, methods to regulate membrane fouling by blocking microbial quorum sensing, i.e., quorum quenching (QQ), have emerged. One promising technology for delaying membrane fouling is the use of quorum quenching bacteria to regulate bacterial biofilm formation. This technology has been widely studied due to its advantages, including the elimination of chemical reagents, no damage to membrane components, full utilization of microbial characteristics, and the potential to control membrane fouling at its source. However, high-performance native quorum quenching bacteria strains are currently scarce.

[0003] Therefore, screening for Pseudomonas bacteria with highly efficient quorum quenching activity is of great significance for the control of membrane fouling, biofilm inhibition, and environmental microbial regulation. Summary of the Invention

[0004] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a Pseudomonas bacillus, its isolation method, and its application.

[0005] The technical solution adopted in this application is:

[0006] The first aspect of the present invention provides a Pseudomonas bacterium, namely Pseudomonas knackmussii HITSZ-Q1, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC NO: 66162 and deposit date of April 18, 2025.

[0007] Furthermore, the 16S rDNA sequence of the Pseudomonas is shown in SEQ ID NO.1.

[0008] A second aspect of the present invention provides a method for isolating the above-mentioned Pseudomonas bacteria, comprising the following steps:

[0009] S1. Enrichment: An enrichment medium was prepared using N-acylhomoserine lactone as the sole carbon and nitrogen source. The sludge sample was added to the enrichment medium for enrichment culture. After the first enrichment, the enriched bacterial solution was transferred to a new enrichment medium at a volume fraction of 3%–8% and cultured for another 3–6 cycles.

[0010] S2. Isolation: Dilute the bacterial solution obtained from the last enrichment culture and spread it on LB medium to obtain the purified strain;

[0011] S3. Screening: The purified strain was co-cultured with N-acylhomoserine lactone, and its quenching activity was detected. The strain with the best quenching effect was selected as the target strain.

[0012] Further, the N-acylhomoserine lactone is at least one of C4-HSL, C6-HSL, C8-HSL, C12-HSL, 3-oxo-C6-HSL, and 3-oxo-C12-HSL.

[0013] The signaling molecules C4-HSL, C6-HSL, C8-HSL, C12-HSL, 3-oxo-C6-HSL, and 3-oxo-C12-HSL mentioned in this specification are, respectively, N-Butyryl-L-Homoserine Lactone (C4-HSL), N-Hexanoyl-L-Homoserine Lactone (C6-HSL), N-Octanoyl-L-Homoserine Lactone (C8-HSL), N-Dodecanoyl-L-Homoserine Lactone (C12-HSL), and N-(3-Oxohexanoyl)-L-Homoserine Lactone (C12-HSL). At least one of Lactone (3-oxo-C6-HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (N-(3-Oxododecanoyl)-L-Homoserine Lactone (3-oxo-C12-HSL).

[0014] Furthermore, the method for detecting quenching activity in step S3 is a colorimetric method.

[0015] Preferably, the colorimetric method is the Chromobacterium violaceum CV026 colorimetric method.

[0016] A third aspect of the present invention provides an immobilized carrier comprising the aforementioned Pseudomonas bacteria.

[0017] The immobilization carrier is obtained by embedding Pseudomonas aeruginosa in polyvinyl alcohol-sodium alginate dual-network gel microspheres, and the crosslinking agent includes saturated boric acid, 4% CaCl2 solution and 0.5M Na2SO4 solution.

[0018] A fourth aspect of the present invention provides the application of the above-described Pseudomonas or the above-described immobilized carrier in wastewater treatment.

[0019] Furthermore, the application is for membrane fouling control and / or biofilm inhibition.

[0020] Furthermore, the aforementioned membrane is a biofilm formed by Pseudomonas aeruginosa.

[0021] The fifth aspect of the present invention provides a method for wastewater treatment, wherein the above-mentioned Pseudomonas bacteria or the above-mentioned immobilized carrier are added to a membrane bioreactor to achieve membrane fouling control and / or biofilm inhibition.

[0022] The beneficial effects of this application are: the screened strain has good and broad-spectrum quenching activity, and it produces intracellular homoserine lactone acylase quenching signal molecules. Through co-culture, it can effectively reduce the biofilm-forming model bacteria Pseudomonas aeruginosa PAO1 and PA14 (Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa PA14). The concentration of signal molecules in the culture medium and the amount of biofilm formation show that this strain has a strong potential to resist biofilm formation. Attached Figure Description

[0023] Figure 1 The concentration of C6-HSL remaining in the supernatant after five enrichment cycles of the mud sample.

[0024] Figure 2 The degradation rate of C6-HSL by Q1-Q6 bacterial suspension.

[0025] Figure 3 Observation of the morphology of the strain on LB plates.

[0026] Figure 4 SEM images of Q1 bacteria at different magnifications; (a) 2.5 × 10⁻⁶ 3 (a) 1.0 × 10 4 (c) 3 × 10 4 times; (d)5×10 4 times.

[0027] Figure 5 This is a phylogenetic tree of the strain.

[0028] Figure 6 The quenching effect of Q1 bacteria on different signal molecules.

[0029] Figure 7 Quenching activity of intracellular and extracellular components of Q1 bacteria; (a) C8-HSL; (b) 3-oxo-C12-HSL.

[0030] Figure 8 For the acidification and recovery of signal molecules.

[0031] Figure 9 For LC-MS intermediate identification: (a) initial m / z = 50-250; (b) initial m / z = 101.5-102.5; (c) final m / z = 50-250; (d) final m / z = 101.5-102.5.

[0032] Figure 10 (a) Standard substance; (b) Intermediate product.

[0033] Figure 11 The concentrations of signaling molecules in the solitary and co-culture systems; (a) C4-HSL; (b) 3-oxo-C12-HSL.

[0034] Figure 12 The relative biofilm content of Q1 bacteria in co-culture; (a) PAO1; (b) PA14.

[0035] Figure 13 The images show actual Q1 bacteria immobilized vector microspheres. The left image shows the blank control microspheres, and the right image shows the immobilized microspheres (HEB) containing Q1 bacteria.

[0036] Figure 14 This is a schematic diagram illustrating the application of Q1 bacteria immobilized carriers in a membrane bioreactor system.

[0037] Figure 15 This is a graph showing the change in transmembrane pressure difference (TMP) during the operation of a membrane bioreactor system. Detailed Implementation

[0038] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0039] In this specification, the term "Pseudomonas knackmussii HITSZ-Q1" or simply "Q1 bacterium" refers to a type of Pseudomonas belonging to the genus Pseudomonas, preliminarily identified as Pseudomonas knackmussii. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC 66162.

[0040] In this invention, the Q1 bacteria are preserved using the glycerol preservation method. A glycerol:water solution of 1:1 is prepared. 1 mL of Q1 bacterial culture and 1 mL of the prepared glycerol solution are added to a 2 mL bacterial culture tube, mixed thoroughly, and then stored at -80°C. Before use, to activate the bacteria, the culture tube is removed from the refrigerator. After the bacterial culture has thawed, 1 mL of the culture is added to 100 mL of LB liquid medium and activated overnight at 30°C and 180 rpm.

[0041] According to a specific embodiment of the present invention, the 16S rDNA sequencing results of the Q1 bacteria are shown in SEQ ID NO.1:

[0042] SEQ ID NO.1:

[0043]

[0044] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0045] In the embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0046] Example 1: Isolation and Identification of Population Sudden Sterilization

[0047] (1) Enrichment

[0048] Enrichment media using the AHL-type signaling molecule C6-HSL as the sole carbon and nitrogen source were prepared by adding 1 mL of sludge from Futian Water Purification Plant, Nanshan Water Purification Plant, Xiaping Environmental Park Landfill Leachate Treatment Plant, and Dasha River sediment to the media. After culturing at 30℃ and 180 rpm for 2 days, the culture was transferred at a 5% ratio to a new inorganic salt medium. After the second cycle, the enrichment medium from the Xiaping Environmental Park Landfill Leachate Treatment Plant did not show significant turbidity, indicating that this bacterial group had low utilization efficiency of C6-HSL, and therefore it was discarded. For the remaining samples, after five cycles, the supernatant was collected, and the remaining C6-HSL in the supernatant was semi-quantitatively analyzed using a biochromogenic method. Figure 1 As shown, the center point indicates the starting position of the colorimetric reaction. The larger the surrounding purple circle, the more C6-HSL remains, indicating a lower quenching activity. The enriched bacterial community from the Nanshan Water Purification Plant exhibited the highest quenching activity and was selected as the target for subsequent separation and screening.

[0049] The LB medium formulation (g / L) was: tryptone 10, yeast extract 5, sodium chloride 10, with an additional 2% technical agar added to the solid medium. The enrichment medium formulation (g / L) was: C6-HSL: 0.5, NaCl: 1, KCl: 0.5, MgCl2: 0.4, CaCl2: 0.1, Na2SO4: 0.15, KH2PO4: 2, Na2HPO4: 2.25, FeCl3: 0.001, MnCl2: 0.1, ZnCl2: 0.046, with the pH adjusted to approximately 5.5 using HCl. The C6-HSL concentration was semi-quantitatively determined using the Chromobacterium violaceum CV026 chromogenic method. Activated CV026 was cultured in LB medium until OD... 600Approximately 1.0 μL of CV026 bacterial suspension was added to LB solid medium cooled to approximately 50°C at a volume ratio of 10:1 to prepare a colorimetric plate. After the plate solidified, wells were punched, and 50 μL of the sample to be tested was added. The plate was then incubated upright at 30°C for 24 hours. Quantification was based on the colorimetric diameter. The semi-quantitative standard curve was: y = 0.06e 1.278x R 2 =0.992. Quenching rate (%) = (Initial signal molecule concentration - Signal molecule concentration at a certain time point) / Initial signal molecule concentration × 100%.

[0050] (2) Separation

[0051] The bacterial culture was diluted with PBS, and 20 μL was spread onto LB solid medium. The dilution gradient for spreading was 10. -5 After culturing for 24 hours, single colonies with significant differences in morphology and size were picked and streaked onto LB agar plates for purification three times. These colonies were then inoculated into LB medium and incubated overnight. The plates were then washed three times with 50 mM Tris-HCl buffer (pH 7.0) and resuspended. OD was adjusted. 600 To approximately 1.0, add C6-HSL to the bacterial suspension at a final concentration of 10 μM / L (the stock solution of the signal molecule C6-HSL was prepared using acetonitrile as the solvent, with a concentration of 25 mM. Calculate the addition volume based on the required final concentration and add it to the bacterial suspension proportionally to achieve a final C6-HSL concentration of 10 μM). Incubate at 30℃ and 180 rpm for 8 hours, then collect the supernatant. Use the CV026 colorimetric method for semi-quantitative detection to determine the quenching activity of the strains. The quenching activities of the six obtained strains are as follows: Figure 2 As shown, Q1 bacteria exhibited the highest quenching activity within 8 hours, reaching 60.54%.

[0052] The above PBS formulation is as follows (g / L): KH2PO4: 0.27, Na2HPO4: 1.42, NaCl: 8, KCl: 0.2.

[0053] (3) Identification

[0054] The Q1 strain was physiologically and biochemically identified using the Huankai physiological and biochemical identification kit. The results are shown in Table 1. The LB plate morphology and scanning electron micrographs of the Q1 strain were analyzed; the LB plate morphology is shown in the table below. Figure 3 As shown, the colonies are milky white, round, with regular edges, smooth surfaces, and are easily picked up. The scanning electron microscope morphology is as follows: Figure 4As shown, under a 50,000x electron microscope, the single bacterium appears as a short rod-shaped organism with slightly pointed ends, a rough surface, and irregular wrinkles. The bacterial species was identified by 16S rDNA sequencing, performed by Shanghai Sangon Biotech Co., Ltd., and its sequence is shown in SEQ ID NO.1. Sequence alignment using the NCBI database confirmed that Q1 belongs to the genus *Pseudomonas*, presumably *Pseudomonas kirchiri*, and named *Pseudomonas sknackmussii* HITSZ-Q1. The sequenced nucleotide sequence was uploaded to the NCBI database (accession number PQ517097), and a phylogenetic tree of Q1 was constructed using MEGA 7 software, as shown below. Figure 5 As shown. The strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC 66162.

[0055] Table 1. Physiological and biochemical identification results of strain Q1

[0056] VP + Gelatin liquefaction - Methyl red - maltose + Xylose + Mannitol - Acetamide - Arginine dihydrolase + Nitrate reduction + Simon's citrate + DNA -

[0057] Example 2: Suddenness Characteristics and Mechanisms of Strains

[0058] (1) Quenching characteristics

[0059] Quenching activity tests were conducted on short-chain C6-HSL, medium-chain N-octanoyl-L-homoserine lactone (C8-HSL), and long-chain N-(3-oxo-dodecanoyl)-L-homoserine lactone with carbonyl substitution (3-oxo-C12-HSL), respectively. Figure 6 As shown in the figure. The results indicate that although the Q1 strain was selected using C6-HSL as the sole carbon and nitrogen source, it can quench a variety of signal molecules. Its quenching effect on medium- and long-chain signal molecules is far superior to that on short chains. Specifically, Q1 can completely degrade C8-HSL and 3-oxo-C12-HSL at an initial concentration of 200 μg / L within 2 hours, and the degradation rate of C6-HSL reaches approximately 75% within 12 hours. Quantification of signal molecules was performed using LC-MS / MS (AB SCIEX Triple Quad 5500+, column: Waters UPLCBEH C18, 1.7 μm, 2.1 mm × 100 mm) in MRM mode. The injection volume was 1 μL, the injection flow rate was 0.2 mL / min, the column temperature was 35 °C, and the mobile phase was 0.1% formic acid and acetonitrile in a ratio of 45%:55%.

[0060] (2) Identification of intracellular / extracellular enzymes

[0061] To investigate whether the quorum quenching enzyme produced by Q1 bacteria is an intracellular or extracellular enzyme, the quorum quenching activity of the supernatant and bacterial suspension of overnight cultured Q1 bacteria was measured. Figure 7As shown, blank LB medium and thermo-inactivated bacterial suspension had almost no degradation effect on the two signaling molecules. The extracellular supernatant showed a 2-hour degradation rate of 19.4% and 66.7% for C8-HSL and 3-oxo-C12-HSL, respectively, while the bacterial suspension showed a 2-hour degradation rate of 98.9% and 100% for C8-HSL and 3-oxo-C12-HSL, respectively. This indicates that the degradation of signaling molecules by Q1 bacteria mainly occurs intracellularly, primarily producing intracellular quorum quenching enzymes.

[0062] (3) Types of quenching enzymes

[0063] Based on their function, quenching enzymes can be mainly classified into three categories: homoserine lactoneases, homoserine acyltransferases, and homoserine oxidoreductases. Lactoneases function by hydrolyzing and opening the lactone ring, but this reaction is reversible. Under strongly acidic conditions, the lactone ring undergoes repair and reduction, thus restoring activity. To investigate whether the quenching enzymes produced by Q1 bacteria belong to homoserine lactoneases, lactone ring repair was performed using an acidification recovery method. The bacterial suspension after quenching was collected, and the cells were sonicated in an ice-water bath (3 seconds sonication, 3 seconds pause, 200W, 5 minutes). After centrifugation, an appropriate amount of the supernatant was taken and the pH was adjusted to approximately 2.0 with 1M HCl. After overnight incubation, the cells were dried under nitrogen and reconstituted with the same volume of acetonitrile (the blank control was prepared by adjusting the pH of the Tris-HCl buffer solution to approximately 10.0 to simulate the hydrolysis of signal molecules under lactonease action; the rest of the process was the same as the experimental group). The concentrations of signal molecules before and after acidification recovery were measured to determine whether lactonease was involved in the quenching of signal molecules. Depend on Figure 8 It was found that C8-HSL was not detected in the bacterial culture after quenching. After acidification, only 0.31% of C8-HSL was recovered, while 78.6% of the signal molecules were recovered in the control group. Therefore, the major role of lactonease in the degradation of signal molecules by Q1 bacteria can be ruled out.

[0064] To further investigate the quenching enzyme type, the intermediate products accumulated during the quenching process were identified using LC-MS. First, a full scan of the mass-to-charge ratio (m / z) range of 50-250 was performed using primary mass spectrometry. The total ion chromatograms before and after quenching are shown below. Figure 9 As shown in (a) and (b), the retention time of C8-HSL was 3.28 min. Due to the high baseline of the total ion chromatogram, no significant accumulation of intermediate products was found when comparing the total ion chromatograms before and after degradation. However, the products differed after the action of different quenching enzymes, and the mass-to-charge ratio range of possible intermediate products could be extracted from the total ion chromatogram, as shown in Table 2. After extracting the total ion chromatogram based on the possible mass-to-charge ratio range of intermediate products, it was found that product accumulation occurred only in the range of 101.5-102.5 during the degradation of C8-HSL, as shown in Table 2. Figure 9As shown in (c) and (d), the product has a mass-to-charge ratio of 102.1 and a retention time of 1.12 min, suggesting it is a homoserine lactone ring. To verify this hypothesis, a secondary mass spectrometry scan was performed on this product with a mass-to-charge ratio of 102.1, and the results were compared with the secondary mass spectrometry scan of the homoserine lactone ring standard. Figure 10 As shown, the main fragments of the secondary mass spectra of the standard substance and the intermediate product are almost identical, which suggests that the intermediate product is a homoserine lactone ring. This further indicates that homoserine lactone acylase plays a major role in the quenching of signal molecules by Q1 bacteria.

[0065] Table 2 Extraction range of intermediate product material-to-charge ratio

[0066] <![CDATA[[M+H + ]]]> 245.5-246.5 101.5-102.5 144.5-145.5 242.5-243.5

[0067] Example 3 Biomembrane Inhibition

[0068] To verify the inhibitory effect of the selected population quenching bacteria Q1 on biofilm formation, co-culture systems were established with biofilm-forming model bacteria *Pseudomonas aeruginosa* PAO1 and PA14 in shake flasks and 24-well plates, respectively. PAO1, PA14, and Q1 were cultured overnight in LB medium, washed three times with PBS, resuspended, and the OD600 was adjusted to approximately 0.8. Q1, PAO1 / PA14, and LB medium were mixed in a volume ratio of 1:1:98 in both 24-well plates and Erlenmeyer flasks, with a total volume of 500 μL. After static incubation for 24 h, the biofilm formation in the 24-well plates and the concentration of signal molecules in the shake flasks were measured.

[0069] Signal molecules in the bacterial culture were extracted using solid-phase extraction (SPE). The cultured bacterial culture was centrifuged at 8000 rpm for 10 min, and 50 mL of the supernatant was collected as the extraction solution. Heptanyl-gaussine lactone (C7-HSL) at a final concentration of 10 μg / L was added as an internal standard. First, the C18 extraction column (500 mg, 6 mL) was activated with 5 mL methanol and 5 mL ultrapure water. The supernatant was then loaded into the SPE column, and extraction was performed dropwise. After extraction, 10 mL of 10% methanol solution was added to wash away interfering substances, followed by 5 mL of acetonitrile to elute the target molecule from the SPE column. The eluent was dried under nitrogen, and then 1 mL of acetonitrile was added. The eluent was stored at -80℃ until analysis. The results are as follows: Figure 11 As shown, when PAO1 and Q1 were co-cultured, the C4-HSL and 3-oxo-C12-HSL in the culture medium decreased by 41.9% and 49.9% respectively compared with the culture alone. When PA14 and Q1 were co-cultured, the C4-HSL and 3-oxo-C12-HSL in the culture medium decreased by 30.2% and 36.2% respectively compared with the culture alone.

[0070] Biofilm quantification was performed using crystal violet staining. The bacterial culture in the wells was discarded, and the suspended bacteria were gently washed with PBS buffer. The wells were then placed in a 60°C oven for 30 min to fix the biofilm. The fixed biofilm was stained with 100 μL of 0.1% crystal violet solution in the dark. After half an hour, the stain was discarded and excess dye was washed off. Finally, the remaining dye was extracted with 200 μL of 95% ethanol, transferred to new wells, and the OD was measured using a microplate reader. 595 The relative content of biofilm was obtained. For example... Figure 12 As shown, in pure culture, the biofilm formation of Q1 bacteria was approximately 50% of that of PAO1 and PA14, indicating that its own biofilm formation was relatively low. In co-culture, the addition of Q1 bacteria significantly inhibited biofilm formation, reducing the biofilm formation of PAO1 and PA14 by 27.9% and 31.9%, respectively, demonstrating a strong potential to resist biofilm formation.

[0071] Example 4: Preparation and Application of Immobilized Carrier

[0072] (1) Preparation of immobilized carrier

[0073] The immobilized microspheres were prepared using a drop-feed method: 10g of polyvinyl alcohol and 1g of sodium alginate were dissolved in 100mL of pure water, thoroughly dissolved and sterilized in an autoclave, and allowed to cool. 2mL of Q1 bacterial solution with a concentration of 200mg / mL was added to 18mL of the above solution, thoroughly mixed, and then aspirated into a syringe. A saturated boric acid-4% calcium chloride solution was dripped dropwise from a certain height. After pre-crosslinking for 1 hour, the microspheres were removed, washed with ultrapure water, and transferred to 0.5M Na2SO4 solution for further crosslinking for 8 hours. They were then washed with ultrapure water and stored at 4℃ for later use, named HEB (HITSZ-Q1 Entrapping Beads). The empty microspheres were prepared using the same steps except that the 2mL of bacterial solution was replaced with 2mL of PBS. The appearance of the microspheres was as follows. Figure 13 As shown.

[0074] (2) Reactor operation and carrier application

[0075] Two MBRs were built, as shown in the diagram. Figure 14 As shown, they are denoted as R1 and R2, respectively. The membrane module in the reactor is a polyvinylidene fluoride (PVDF) hollow fiber membrane with a membrane area of ​​0.04 m². 2The pore size is 0.22 μm, and the membrane fiber length is 120 mm. Specific operating parameters and carrier dosage are shown in Table 3. Reactor operation is divided into two stages: the first stage is the conventional operation stage, and the second stage is the carrier addition stage. Empty spheres are added to R1, and HEB is added to R2. The transmembrane pressure difference is used to indicate the degree of membrane fouling. Under constant flow rate operation, the value indicates the degree of membrane module fouling; a higher value indicates more severe membrane fiber fouling. When the transmembrane pressure difference rises to 35 kPa, the membrane module is considered completely fouled and requires cleaning and regeneration, at which point the operating cycle ends. The membrane module is removed and soaked in 1 L of pure water for 2 hours, then soaked in a 0.5% sodium hypochlorite solution for 2 hours for chemical cleaning. Finally, the membrane module is repeatedly rinsed with pure water to regenerate it for the next cycle. The transmembrane pressure difference between R1 and R2 during operation is shown in Table 3. Figure 15 As shown in the diagram, during the traditional operation phase, the transmembrane pressure difference changes in the membrane fouling process of R1 and R2 showed similar trends with no significant difference, both reaching 35 kPa at approximately 6.5 days. In the second phase, R1, with empty spheres added, ran for two cycles, each cycle lasting approximately 6 days, indicating that the addition of empty spheres had no significant effect on alleviating membrane fouling. R2, with HEB added, ran for one cycle in this phase, and the transmembrane pressure difference reached approximately 35 kPa at 10.5 days. Compared to the first phase, the membrane fouling delay rate with HEB addition was 61.5%, fully demonstrating that HEB addition can alleviate the MBR membrane fouling rate.

[0076] The simulated wastewater formulation (g / L) is as follows: glucose: 0.3, starch: 0.2, NH4Cl: 0.096, CO(NH2)2: 0.032, KH2PO4: 0.0235, MgSO4·7H2O: 0.02, CaCl2: 0.025, FeSO4·7H2O: 0.0012, MnSO4·H2O: 0.000155, ZnSO4·7H2O: 0.00022, CuSO4: 0.000125. Membrane fouling delay rate (%) = (Membrane fouling cycle after HEB addition - Membrane fouling cycle during traditional operation) / Membrane fouling cycle during traditional operation × 100%.

[0077] Table 3 Reactor operating parameters

[0078] Effective volume 4L Hydraulic residence time 10h sludge retention time No mud was discharged during the experiment. Initial sludge concentration Approximately 6500 mg / L Membrane flux <![CDATA[10L / m 2 ·h]]> Aeration intensity 1.0L / min Carrier dosage 1% (v / v)

[0079] In this embodiment, the carrier dosage is 1% (v / v). In other embodiments, the carrier dosage may be 0.5% to 1.5% (v / v).

[0080] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A type of Pseudomonas, characterized in that, The Pseudomonas species mentioned is *Pseudomonas krusei* (…). Pseudomonas knackmussii The *Pseudomonas* strain HITSZ-Q1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66162; the *Pseudomonas* strain produces intracellular homoserine lactone acylase.

2. The *Pseudomonas* strain according to claim 1, characterized in that, The 16S rDNA sequence of the Pseudomonas is shown in SEQ ID NO.

1.

3. An immobilized carrier, characterized in that, The immobilized carrier contains the Pseudomonas bacteria as described in claim 1 or 2.

4. The application of the Pseudomonas bacillus as described in claim 1 or 2, or the immobilized carrier as described in claim 3, in wastewater treatment; The applications are membrane fouling control and / or biofilm inhibition; The membrane is a biofilm formed by Pseudomonas aeruginosa.

5. A method for wastewater treatment, characterized in that, The method includes the following steps: adding the Pseudomonas aeruginosa as described in claim 1 or 2 or the immobilized carrier as described in claim 3 into a membrane bioreactor to achieve membrane fouling control and / or biofilm inhibition, wherein the membrane is a biofilm formed by Pseudomonas aeruginosa.

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