Strain for urban runoff pollution control and water body eutrophication treatment and application thereof

Abstract

The application relates to a strain for controlling urban runoff pollution and treating water body eutrophication and application thereof, and belongs to the field of microorganisms, in particular to a strain for controlling urban runoff pollution and treating water body eutrophication and application thereof.The strain for controlling urban runoff pollution and treating water body eutrophication is characterized in that the strain is Enterobacter asburiae SW-78, is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 37681.The strain is an Enterobacter asburiae strain with the functions of salt and alkali tolerance, iron carrier production, organic phosphorus degradation and nitrogen fixation, provides a novel microbial technical scheme for controlling urban runoff pollution and treating water body eutrophication, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of microorganisms used for urban runoff pollution control and eutrophication treatment, and its applications. Background Technology

[0002] During urbanization, urban runoff carries large amounts of nitrogen and phosphorus nutrients, organic pollutants, and salts. When these runoffs flow into water bodies, they easily cause eutrophication. Furthermore, both the runoff and the receiving water bodies often exhibit slightly saline-alkaline characteristics (NaCl 100-400 mmol / L, pH 8.0-9.5). Existing treatment technologies have significant shortcomings: physical interception can only remove particulate-bound pollutants; chemical phosphorus removal is prone to secondary pollution and its efficiency decreases in alkaline environments; and most commonly used biological agents are not salt-alkali tolerant, have limited functions, and cannot simultaneously reduce complex pollutants. In addition, iron deficiency inhibits microbial activity, leading to unstable treatment results. Summary of the Invention

[0003] In order to solve the problems described in the background art above, the present invention provides a strain for urban runoff pollution control and eutrophication treatment of water bodies and its application.

[0004] The present invention relates to a strain for urban runoff pollution control and eutrophication treatment, characterized in that the strain is Enterobacter asburiae SW-78, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37681.

[0005] Application of Enterobacter asburiae SW-78 in urban runoff pollution control and eutrophication treatment.

[0006] The bacterial agent used in this invention for urban runoff pollution control and eutrophication treatment includes Enterobacterasburiae SW-78.

[0007] Furthermore, the bacterial agent is a liquid bacterial agent or a powdered bacterial agent.

[0008] The salt- and alkali-tolerant synergistic purification fermentation medium of Enterobacter asburiae SW-78 of this invention comprises 17.0 g / L glucose, 15.0 g / L defatted soybean meal, 6.5 g / L yeast extract, 3.8 g / L ammonium sulfate, 17.5 g / L sodium chloride, 2.6 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate, 0.006 g / L ferrous sulfate, 2.3 g / L humic acid, 1.6 g / L trisodium citrate, 2.8 g / L lecithin, and 0.5 g / L L-proline.

[0009] Enterobacter asburiae, a typical environmental functional bacterium, is widely distributed in habitats such as soil and water sediment. Studies have confirmed its basic potential for nutrient transformation and pollutant degradation, demonstrating its application value in scenarios such as phosphorus activation in agricultural soils and purification of lightly polluted water bodies. However, existing isolates mostly focus on single environmental needs, lacking salt and alkali tolerance and multifunctional synergy, making them difficult to adapt to the complex requirements of municipal environmental governance. The Enterobacter asburiae SW-78 strain obtained in this invention can grow stably in environments with 0-400 mmol / L NaCl and pH 7.0-9.5, producing a siderophore SU value of 78.45%. It also possesses functions of organic phosphorus solubilization and nitrogen fixation. Through "stress tolerance + multifunctional synergy," it achieves simultaneous interception of runoff pollution and treatment of eutrophication, solving the technical problems of poor adaptability and single function of existing bacterial agents, and providing a new type of microbial resource for municipal environmental governance.

[0010] The strain of this invention is an Enterobacterasburiae strain that is salt and alkali tolerant, produces iron carriers, decomposes organic phosphorus, and fixes nitrogen. It provides a novel microbial technology solution for urban runoff pollution control and eutrophication treatment, and has good prospects for industrial application.

[0011] Enterobacter asburiae SW-78 is Enterobacter asburiae, belonging to the genus Enterobacter. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37681 and deposit date of February 3, 2026. Attached Figure Description

[0012] Figure 1 These are colony photos of strain SW-78 obtained in Example 1 after being inoculated on CAS medium and cultured for a period of time;

[0013] Figure 2 These are colony photos of strain SW-78 obtained in Example 1 after being inoculated on Monkina organophosphorus solid medium and cultured for a period of time;

[0014] Figure 3 These are colony photos of strain SW-78 obtained in Example 1 after being inoculated on Assumption medium and cultured for a period of time;

[0015] Figure 4 This is a phylogenetic tree constructed from strain SW-78 obtained in Example 1. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Definitions and explanations:

[0018] SKM medium: Skim Milk Medium, also known as skim milk medium.

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0020] Example 1

[0021] The strain used in this embodiment for urban runoff pollution control and eutrophication treatment is Enterobacterasburiae SW-78, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37681.

[0022] In September 2025, sediment from the Dashahe Wetland Park in Liwan District, Guangzhou City, Guangdong Province, was transported back to the laboratory via cold chain. 5g of sediment was placed in an Erlenmeyer flask containing glass beads and 50 mL of sterile water in a clean bench and shaken at 180 r / min at room temperature for 30 min. Then, a serial dilution was performed, with 10g of sediment added to each flask. -3 10 -4 10 -5 100 μL of each gradient was spread onto LB agar plates, with each gradient repeated three times, and incubated at 28°C for 48 h. After 48 h of incubation, strains with different characteristics were selected for isolation, numbered, and cultured separately.

[0023] Screening of strains with siderophore-producing ability:

[0024] The purified strain was reactivated and transferred to LB agar plates for 24 hours. Single colonies were then picked with sterile toothpicks and inoculated onto Chromeazurol S (CAS) solid detection medium. The plates were incubated upside down at 37°C for 2-3 days. The size of the discoloration zone around the colony was observed. The colony diameter d and the discoloration zone diameter D were measured using the cross-cross method, and the ratio D / d was calculated. Further experiments were conducted on strains exhibiting a clear discoloration zone.

[0025] (1) The activated bacterial motility was inoculated into SA iron-limited medium (liquid medium) and cultured in a shaker at 37 ˚C for 48 h;

[0026] (2) Transfer the bacterial suspension to be tested after 48 hours of growth to a sterilized 10 mL centrifuge tube and centrifuge at 13,000 rpm for 15 min.

[0027] (3) Transfer the supernatant to a test tube treated with concentrated hydrochloric acid, add a certain amount of freshly prepared CAS detection solution to make the volume ratio of supernatant to CAS detection solution 1:1, mix thoroughly and let stand at room temperature for 1 h.

[0028] (4) Measure the absorbance (As) of the above-mentioned standing solution at a wavelength of 630 nm. Use double-distilled water as a control to zero the solution. Use the absorbance (Ar) of the uninoculated SA iron-limiting medium mixed with the CAS detection solution as a reference value. Express the iron carrier activity unit as follows:

[0029] Su≈(Ar-As) / Ar×100;

[0030] In the formula: Su is the iron carrier content; Ar is the OD value of the uninoculated SA iron-limited medium and the CAS detection solution; As is the OD value of the SA iron-limited medium supernatant and the CAS detection solution of the inoculated strain.

[0031] When the number of ferrocarrier activity units is less than 10, it is generally considered negative, and the mixture of ferrocarrier and test solution does not show any color change.

[0032] Screening for strains with organophosphate solubility:

[0033] The isolated and purified strains were inoculated onto plates of Monkina organophosphate bacteria culture medium using sterile toothpicks. Each gradient was repeated three times, and the plates were incubated at 28°C for 24–48 h. Colonies exhibiting organophosphate-solubilizing zones were selected, and the colony diameter d and the diameter D of the organophosphate-solubilizing zone were measured using the cross-crossing method. The D / d ratio was calculated, and strains with high ratios were selected for purification and their organophosphate-solubilizing effects were repeatedly verified.

[0034] Screening for nitrogen-fixing strains:

[0035] The isolated and purified strains were inoculated onto Assumption agar plates using sterile toothpicks. Each gradient was repeated three times, and the plates were incubated at 28°C for 24–48 h. Colonies exhibiting a clear zone were selected, and the colony diameter d and the diameter D of the nitrogen-fixing ring were measured using the cross-crossing method. The D / d ratio was calculated, and strains with high ratios were selected for purification, and their nitrogen-fixing efficiency was repeatedly verified.

[0036] Screening for salt-tolerant strains:

[0037] LB agar plates containing NaCl at concentrations of 50 mmol / L, 100 mmol / L, 200 mmol / L, 300 mmol / L, 400 mmol / L, 450 mmol / L, 500 mmol / L, 550 mmol / L, 600 mmol / L, and 650 mmol / L were prepared. The isolated and purified bacterial strains were inoculated onto LB agar plates using the streak plating method, with a medium without NaCl serving as a control. The plates were sealed and inverted in a constant temperature incubator at 28 ℃ for 24–48 h, during which colony growth was observed.

[0038] Screening for alkali-resistant strains:

[0039] LB solid culture media with pH values ​​of 7-12.5 were prepared separately. The isolated and purified bacterial strains were inoculated onto LB solid culture media using the streak plating method, with the pH 7 medium used as a control. The media were sealed and inverted in a constant temperature incubator at 28 ℃ for 24-48 h, during which colony growth was observed. Further identification was performed based on the colony growth.

[0040] In this embodiment, 832 strains with different morphologies were screened from the sediment. Among them, 6 strains simultaneously exhibited salt and alkali tolerance, iron production, organic phosphorus solubilization, and nitrogen fixation. Strain SW-78, with relatively good overall capabilities, was selected. Strain SW-78 was inoculated onto CAS medium, and after a period of cultivation, a distinct discoloration zone (e.g., ...) formed around the colony. Figure 1 As shown in the figure, the diameter of the discoloration zone D of strain SW-78 is 12.53 mm, the colony diameter d is 2.70 mm, and the D / d ratio is 4.64, indicating that strain SW-78 has a very strong siderophore with high iron chelation ability.

[0041] The Su value of siderophore production by strain SW-78 at 37℃ was 78.45%, indicating that the strain has a very strong siderophore production capacity.

[0042] When strain SW-3 was inoculated onto Monkina organophosphate medium, after a period of cultivation, a distinct zone of dissolved organophosphates (such as...) formed around the colonies. Figure 2 As shown in the figure, the diameter D of the organophosphate solubilization zone of strain SW-3 is 19.04 mm, the colony diameter d is 8.65 mm, and the D / d ratio is 2.20, indicating that strain SW-3 has a strong organophosphate solubilization function.

[0043] When strain SW-17 was inoculated onto Assumption medium, after a period of cultivation, a distinct nitrogen-fixing zone (e.g., ...) formed around the colony. Figure 2As shown in the figure, the nitrogen fixation zone diameter D of strain SW-17 is 5.77 mm, the colony diameter d is 2.10 mm, and the D / d ratio is 2.75, indicating that strain SW-17 has a strong nitrogen fixation function.

[0044] Strain SW-78 can grow normally at NaCl concentrations ranging from 0 to 400 mmol / L, as shown in Table 1, indicating that strain SW-78 has strong salt tolerance.

[0045] Table 1

[0046]

[0047] Note: + indicates growth; - indicates no growth.

[0048] Strain SW-78 can grow normally at pH 9.5, as shown in Table 2, indicating that strain SW-78 has strong alkali resistance.

[0049] Table 2

[0050]

[0051] Note: + indicates growth; - indicates no growth.

[0052] Identification of strain SW-78:

[0053] Strand strain SW-78 was streaked in three zones on a solid LB medium plate, and single colonies were isolated and their morphology was described. Gram staining and physiological and biochemical identification of the strain were performed according to the "Handbook of Common Bacterial System Identification".

[0054] The colony characteristics of strain SW-78 on LB solid medium are as follows: pale yellow, round, raised colonies with a smooth, moist surface, soft texture, and easy to pick up; no water-soluble pigment is produced; and it is identified as a Gram-negative bacterium by Gram staining. Some physiological and biochemical indicators of strain SW-78 are shown in Table 3. Based on the descriptions of the physiological and biochemical characteristics of Enterobacter asburiae in Bergey's Manual of Bacteriology and related literature, strain SW-78 shares the same physiological and biochemical characteristics as the type species of Enterobacter asburiae. Based on these physiological and biochemical indicators, it is inferred that strain SW-78 may be Enterobacter asburiae.

[0055] Table 3

[0056]

[0057] Note: + indicates a positive result; - indicates a negative result.

[0058] Identification of 16S rRNA in strain SW-78:

[0059] DNA from strain SW-78 was extracted and purified using a bacterial genomic DNA extraction kit from Beijing Solarbio Biotechnology Co., Ltd. PCR amplification was performed using universal bacterial primers 27F / 1492R. The PCR amplification system was 25 µL: 2.5 µL 10× buffer, 0.5 µL Taq enzyme, 0.5 µL primer 27F, 0.5 µL primer 1492R, 1 µL DNA template, and 20 µL ddH2O. The reaction program was: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 50 s, 56℃ annealing for 30 s, 72℃ extension for 1.5 min, for 30 cycles; a final extension at 72℃ for 10 min; and storage at 4℃. The PCR products were sent to RuiBiotech for sequencing. The sequencing results of the strain's 16S rRNA were compared with the NCBI database, and a phylogenetic tree was constructed.

[0060] BLAST comparison in NCBI revealed that the 16S rRNA gene sequence of strain SW-78 had a 99% similarity to Enterobacterasburiae. Figure 4 As shown in the phylogenetic tree of strain SW-78, it can be seen that strain SW-78 and Enterobacter asburiae (JM-458) are in the same smallest branch and have a close evolutionary distance. Based on the comprehensive physiological and biochemical indicators, strain SW-78 is identified as Enterobacter asburiae.

[0061] Example 2

[0062] The salt- and alkali-tolerant synergistic purification fermentation medium for Enterobacter asburiae SW-78 consists of: glucose 17.0 g / L, defatted soybean meal powder 15.0 g / L, yeast extract 6.5 g / L, ammonium sulfate 3.8 g / L, sodium chloride 17.5 g / L (300 mmol / L), dipotassium hydrogen phosphate 2.6 g / L, potassium dihydrogen phosphate 1.2 g / L, magnesium sulfate 0.7 g / L, ferrous sulfate 0.006 g / L, humic acid 2.3 g / L, trisodium citrate 1.6 g / L, lecithin (organic phosphorus source) 2.8 g / L, L-proline 0.5 g / L, and deionized water.

[0063] The steps for culturing Enterobacter asburiae SW-78 using the above culture medium are as follows:

[0064] pH adjustment: Adjust to 9.0-9.5 using 1mol / L NaOH solution. After sterilization, the pH stabilizes at 8.8-9.3, requiring no secondary adjustment, which is suitable for the alkali-tolerant characteristics of the strain.

[0065] Sterilization conditions: autoclave at 121℃ for 20 minutes, cool to below 30℃ for aseptic inoculation (avoid high temperature to prevent damage to lecithin and stress protectant activity).

[0066] Inoculation rate: Inoculate with Enterobacter asburiae SW-78 seed culture (OD) at 2% (v / v). 600 =0.8-1.0), shortening the strain's adaptation period to combined stress;

[0067] Fermentation control: temperature 28℃, shaking speed 180rpm (to ensure sufficient dissolved oxygen and adapt to the metabolic needs of the strain), fermentation cycle 48h;

[0068] Endpoint indicator: viable bacteria count ≥ 1.0 × 10⁻⁶ 9 CFU / mL, organophosphorus lyase activity ≥105U / mL, nitrogenase activity ≥92U / mL, siderophore SU value ≥76%, and survival rate of the strain ≥90% in an environment of 400mmol / L NaCl and pH9.5.

[0069] Table 4 shows a comparison of the core parameters of the ordinary LB medium and the salt- and alkali-tolerant synergistic purification fermentation medium of the present invention.

[0070] Table 4 Comparison of core parameters between ordinary LB medium and the salt- and alkali-tolerant synergistic purification fermentation medium of this invention

[0071]

[0072] Example 3

[0073] Seed culture preparation: The refrigerated strain Enterobacter asburiae SW-78 was inoculated into a salt- and alkali-tolerant synergistic purification fermentation medium and cultured at 28°C and 180 rpm for 24 h until the strain reached OD. 600 When the value reaches 0.8-1.0, seed solution is obtained;

[0074] Preparation of liquid inoculum: Inoculate the seed culture at a rate of 1%-3% into a salt- and alkali-tolerant synergistic purification fermentation medium, and culture at 28℃ and 180 rpm for 48 hours until the Enterobacter asburiae SW-78 count in the fermentation broth is ≥1.0 × 10⁻⁶. 9 CFU / mL yields a liquid bacterial agent;

[0075] Targeted application in buffer zones: In the near-shore ecological buffer zone (5-10m wide) of urban water bodies, following the principle of "strip application," dilute the liquid bacterial agent with clean water at a ratio of 1:400, and spray it evenly onto the soil and plant rhizosphere of the buffer zone using a low-pressure sprayer. The application rate is 40-60L / 1000m². 2The dosage around runoff inlets has been increased to 80L / 1000m³. 2 This forms a "pollution interception zone";

[0076] In-situ synergistic purification: Enterobacter asburiae SW-78 colonizes the soil and rhizosphere of plants in the buffer zone, degrades organic phosphorus by decomposing organic pollutants carried by runoff, converts atmospheric nitrogen by fixing nitrogen, and produces iron carriers to promote nutrient absorption by plants. The physical interception and microbial degradation in the buffer zone form a synergistic effect.

[0077] Operation and maintenance: Re-spray diluted Enterobacter asburiae SW-78 liquid bacterial agent every 25 days (the amount of the re-spray is 50% of the initial amount), and add one more re-spray after the flood season (July-August); monitor the water transparency and total phosphorus concentration. When the total phosphorus is ≤0.2mg / L, the frequency of addition can be reduced to adapt to the operation and maintenance cycle of municipal water bodies.

[0078] The comparison results of indicators before and after processing the nearshore ecological buffer zone and landscape water body using the method of this embodiment are shown in Table 5; where n is the number of test samples, n=5, and the data in the table are the average values ​​of the measurements; processing time: continuous operation for 45 days.

[0079] The method described in this embodiment is applicable to nearshore ecological buffer zones of urban rivers, lakes, and landscape water bodies. It is suitable for in-situ treatment of eutrophication and addresses the accumulation of nitrogen and phosphorus in water bodies after runoff inflow, combined with enhanced purification through buffer zones.

[0080] Table 5 Comparison of indicators before and after treatment of nearshore ecological buffer zones and landscape water bodies with liquid bacterial agents.

[0081]

[0082] Example 4

[0083] Seed culture preparation: Cold-preserved Enterobacter asburiae SW-78 was inoculated into a salt- and alkali-tolerant synergistic purification fermentation medium and cultured at 28°C and 180 rpm for 24 hours until the strain reached OD500. 600 When the value reaches 0.8-1.0, seed solution is obtained;

[0084] Preparation of liquid inoculum: Inoculate the seed culture at a rate of 1%-3% into a salt-tolerant and alkali-resistant synergistic purification fermentation medium, and culture at 28℃ and 180 rpm for 48 hours until the viable count of Enterobacter asburiae SW-78 in the fermentation broth is ≥1.0×10⁻⁶. 9 CFU / mL yields a liquid bacterial agent;

[0085] Preparation of powdered microbial agent: Enterobacter asburiae SW-78 liquid microbial agent (viable count ≥ 1.0 × 10⁻⁶) 9 A mixture of CFU / mL of sterile diatomaceous earth and well-rotted straw powder (mass ratio 3:2) was vacuum dried until the moisture content was ≤15% to obtain a powdered bacterial agent. The viable count of Enterobacter asburiae SW-78 was ≥1.0×10⁻⁶. 8 CFU / g, the powder is easy to disperse and settles quickly, making it suitable for the mixing conditions of regulating tanks;

[0086] Addition and operation of the stormwater storage tank: An automatic dosing device is installed at the inlet of the stormwater storage tank. Powdered bacterial agent is added at a ratio of 0.2%-0.3% of the inflow rate. The mixture is stirred in the storage tank for 15-20 minutes using a stirring device (50-80 rpm), with a retention time of 4-6 hours. The Enterobacter asburiae SW-78 strain is rapidly activated in the water, simultaneously degrading pollutants through organophosphate decomposition and nitrogen fixation, and producing iron carriers to enhance the synergistic effect.

[0087] Pre-treatment and disposal: After pre-treatment, the rainwater is settled in the regulating tank (2-3 hours). The supernatant is discharged into the urban pipe network or directly replenishes the water body. The bottom sludge can be discharged periodically (once a month) as raw material for organic fertilizer for landscaping (the strains are non-pathogenic and there is no secondary pollution).

[0088] Large-scale operation and maintenance: Adjust the parameters of the dosing device according to the frequency of rainfall. Automatic dosing is turned on during the rainy season (June-September) and turned off during the dry season. Check the storage of microbial agent every 15 days and replenish Enterobacter asburiae SW-78 powder microbial agent. It is compatible with the existing dredging and mixing system of the storage tank and requires no additional equipment modification.

[0089] The comparison results of indicators before and after treating the runoff of the rainwater storage tank using the method of this embodiment are shown in Table 6; where n is the number of test samples, n=5, and the data in the table are the average values ​​of the measurements; the treatment time is 5 hours for a single stay and 30 days for continuous operation.

[0090] The method described in this embodiment is applicable to urban stormwater storage tanks (centralized runoff pretreatment facilities), and is suitable for large-scale runoff pollution pretreatment. It reduces nitrogen, phosphorus and organic pollutants before rainwater enters the water body, thereby reducing the pressure on subsequent water body treatment.

[0091] Table 6 Comparison of runoff indicators before and after treatment with powdered microbial agents in rainwater storage tanks

[0092]

Claims

1. A bacterial strain for urban runoff pollution control and eutrophication treatment, characterized in that, The strain described is Enterobacter asburiae SW-78, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37681.

2. The application of the strain described in claim 1 in urban runoff pollution control and eutrophication treatment.

3. A microbial agent for urban runoff pollution control and eutrophication treatment, characterized in that, The bacterial agent includes Enterobacter asburiae SW-78.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent is a liquid bacterial agent or a powdered bacterial agent.

5. A salt- and alkali-tolerant synergistic purification fermentation medium for Enterobacter asburiae SW-78, characterized in that the medium comprises 17.0 g / L glucose, 15.0 g / L defatted soybean meal, 6.5 g / L yeast extract, 3.8 g / L ammonium sulfate, 17.5 g / L sodium chloride, 2.6 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate, 0.006 g / L ferrous sulfate, 2.3 g / L humic acid, 1.6 g / L trisodium citrate, 2.8 g / L lecithin, and 0.5 g / L L-proline.

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