Strain for remediation of organic pollution in urban green soil and application thereof

Abstract

The application relates to a strain for repairing organic pollution of urban green land soil and application thereof, and belongs to the field of microorganisms, in particular to a strain for repairing organic pollution of urban green land soil and application thereof.The strain for repairing organic pollution of urban green land soil is Priestia megaterium XG-175, which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No.38209.The strain has the functions of alkali tolerance, iron carrier production, protein degradation, organic phosphorus degradation and cellulose degradation, provides a novel microbial technical scheme for repairing organic pollution of urban green land soil, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain for the remediation of organic pollution in urban green space soil and its application. Background Technology

[0002] As a core component of the urban ecosystem, urban green spaces are susceptible to the accumulation of fallen leaves and branches, as well as sewage and organic waste carried by surface runoff. This leads to the accumulation of pollutants such as cellulose macromolecular organic matter, proteins, and organophosphates in the soil. These pollutants not only reduce soil permeability and fertility but may also infiltrate groundwater with rainwater. Furthermore, some urban green space soils are slightly alkaline (pH 8.0-11.0) due to irrigation water and geological background. This alkaline environment inhibits the activity of native microorganisms, exacerbating the retention of organic pollution and becoming a core challenge in the ecological maintenance of urban green spaces.

[0003] Currently, the remediation of organic pollution in urban green space soil mainly employs methods such as physical tillage, application of chemical degrading agents, and application of single-function microbial agents. Physical tillage is slow to take effect and easily damages soil structure; chemical degrading agents pose a risk of secondary pollution and cannot target the degradation of complex organic pollutants; biological methods have become mainstream due to their environmental friendliness, but existing microbial agents have significant drawbacks: most microbial agents have a single function, only degrading cellulose or solubilizing phosphorus, and cannot simultaneously treat multiple organic pollutants in the soil; some multifunctional microbial agents lack the ability to produce iron carriers, and the scarcity of available iron in the soil limits the metabolic activity and remediation efficiency of the strains; moreover, they lack alkali tolerance, making them difficult to adapt to alkaline green space soils, thus limiting their application scenarios. Summary of the Invention

[0004] In order to solve the problems described in the background art above, the present invention provides a strain for the remediation of organic pollution in urban green space soil and its application.

[0005] The strain used in this invention for the remediation of organic pollution in urban green space soil is Priestia megaterium XG-175, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38209.

[0006] Application of Priestia megaterium XG-175 in the remediation of organic pollution in urban green space soil.

[0007] The microbial agent used in this invention for the remediation of organic pollution in urban green space soil includes Priestia megaterium XG-175.

[0008] Furthermore, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0009] The alkali-tolerant synergistic fermentation medium for Priestia megaterium XG-175 of this invention comprises 18.0 g / L glucose, 22.0 g / L defatted soybean meal, 10.0 g / L sodium carboxymethyl cellulose, 6.0 g / L yeast extract, 5.0 g / L ammonium sulfate, 3.0 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, 0.3 g / L calcium chloride, 0.009 g / L ferrous sulfate, 2.5 g / L humic acid, and 2.0 g / L urea.

[0010] Priestia megaterium is a typical stress-resistant functional bacterium. Although it has been proven to have certain enzyme production and nutrient conversion capabilities, none of the existing isolates have been found to possess the quadruple functions of degrading organophosphates, producing cellulase, producing siderophores, and being alkali-tolerant. This invention screened Priestia megaterium strain XG-175 from soil. This strain can grow stably in alkaline soils with a pH of 7.0-9.8. It simultaneously degrades multiple organic pollutants in the soil through degrading organophosphates and producing cellulase. Its siderophore production (SU value 77.93%) enhances microbial activity. This solves the technical problems of existing bacterial agents having single functions, poor alkali tolerance, and low remediation efficiency, providing a novel microbial resource for the remediation of organic pollution in urban green space soils.

[0011] The strain of this invention is a Priestiamegaterium strain that combines alkali tolerance, iron carrier production, protein solubilization, organophosphate solubilization, and cellulose solubilization functions. It provides a novel microbial technology solution for the remediation of organic pollution in urban green space soil and has good prospects for industrial application.

[0012] Priestia megaterium XG-175 is a species of Priestia megaterium, belonging to the genus Priestia. 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. 38209 and deposit date of February 3, 2026. Attached Figure Description

[0013] Figure 1 These are colony photos of strain XG-175 obtained in Example 1 after being inoculated on CAS medium and cultured for a period of time;

[0014] Figure 2 These are colony photos of strain XG-175 obtained in Example 1 after being inoculated on SKM medium and cultured for a period of time;

[0015] Figure 3These are colony photos of strain XG-175 obtained in Example 1 after being inoculated on Monkina organophosphate solid medium and cultured for a period of time;

[0016] Figure 4 These are colony photos of strain XG-175 obtained in Example 1 after being inoculated on cellulose Congo red medium and cultured for a period of time;

[0017] Figure 5 This is a phylogenetic tree constructed from strain XG-175 obtained in Example 1. Detailed Implementation

[0018] 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.

[0019] Definitions and explanations:

[0020] SKM medium: Skim Milk Medium, also known as skim milk medium.

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0022] Example 1

[0023] The strain used in this embodiment for the remediation of organic pollution in urban green space soil is Priestia megaterium XG-175, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38209.

[0024] In October 2025, surface soil from the Yongding Riverside Park in Shougang Park, Shijingshan District, Beijing, was transported back to the laboratory via cold chain. 5g of the soil 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, resulting in 10... -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.

[0025] Screening of strains with siderophore-producing ability:

[0026] 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.

[0027] (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;

[0028] (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.

[0029] (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.

[0030] (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:

[0031] Su≈(Ar-As) / Ar×100;

[0032] 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.

[0033] 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.

[0034] Screening for strains with protein-degrading capabilities:

[0035] The isolated and purified bacterial strains were inoculated onto SKM (2% skim milk powder) medium 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 protein-lysing zone were selected, and the colony diameter d and the protein-lysing zone diameter D were measured using the cross-crossing method. The D / d ratio was calculated, and strains with the highest ratios were selected for purification, and their protein-dissolving effect was repeatedly verified.

[0036] Screening of strains with cellulose-degrading ability:

[0037] The isolated and purified strains were inoculated onto plates of cellulose-containing Congo red bacteria agar using sterile toothpicks. Each gradient was repeated three times, and the plates were incubated at 28°C for 24–48 h. Colonies exhibiting cellulose-dissolving zones were selected, and the colony diameter d and the diameter D of the cellulose-dissolving 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 cellulose-dissolving effect was repeatedly verified.

[0038] Screening for strains with organophosphate solubility:

[0039] 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.

[0040] Screening for alkali-resistant strains:

[0041] 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.

[0042] In this embodiment, 482 strains with different morphologies were screened from the soil. Among them, 8 strains simultaneously exhibited alkali tolerance, siderophore production, protein solubilization, organophosphate solubilization, and cellulose solubilization. Strain XG-175, with relatively good overall capabilities, was selected. Strain XG-175 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 D of the discoloration zone of strain XG-175 is 14.68 mm, the colony diameter d is 3.24 mm, and the D / d ratio is 4.53, indicating that strain XG-175 has a strong siderophore capable of producing high iron chelation.

[0043] The Su value of siderophore production by strain XG-175 at 37℃ was 77.93%, indicating that the strain has a strong siderophore production capacity.

[0044] When strain XG-175 was inoculated onto SKM medium, after a period of incubation, a distinct deproteinization zone (e.g., ...) formed around the colony. Figure 2 As shown in the figure, the diameter of the protein-degrading zone D of strain XG-175 is 13.75 mm, the colony diameter d is 5.70 mm, and the D / d ratio is 2.41, indicating that strain XG-175 has a strong protein-degrading function.

[0045] When strain XG-175 was inoculated onto Monkina organophosphate medium, after a period of cultivation, a distinct zone of dissolved organophosphates (such as...) formed around the colonies. Figure 3 As shown in the figure, the diameter D of the organophosphate solubilization zone of strain XG-175 is 24.28 mm, the colony diameter d is 18.50 mm, and the D / d ratio is 1.31, indicating that strain XG-175 has a strong organophosphate solubilization function.

[0046] When strain XG-175 was inoculated onto cellulose Congo red medium, after a period of cultivation, a distinct decellulose-degrading zone formed around the colony (e.g., Figure 4 As shown in the figure, the diameter of the cellulose-degrading zone D of strain XG-175 is 9.44 mm, the colony diameter d is 2.44 mm, and the D / d ratio is 3.87, indicating that strain XG-175 has a strong cellulose-degrading function.

[0047] Strain XG-175 can grow normally at pH 9.8, as shown in Table 1, indicating that strain XG-175 has strong alkali resistance.

[0048] Table 1

[0049]

[0050] Note: + indicates growth; - indicates no growth.

[0051] Identification of strain XG-175:

[0052] Strand strain XG-175 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".

[0053] The colony characteristics of strain XG-175 on LB solid medium are as follows: milky yellow, round, with neatly defined raised colonies; smooth, moist surface without wrinkles; soft and loose texture; no stringy formation when picked up with an inoculation loop; no water-soluble or fat-soluble pigments produced; and Gram-positive bacteria identified by staining. Some physiological and biochemical indicators of strain XG-175 are shown in Table 2. Based on the descriptions of the physiological and biochemical characteristics of *Priestia megaterium* in Bergey's Manual of Bacteriology and related literature, strain XG-175 shares the same physiological and biochemical characteristics as the type species *Priestia megaterium*. Therefore, based on these physiological and biochemical indicators, strain XG-175 is likely *Priestia megaterium*.

[0054] Table 2

[0055]

[0056] Note: + indicates a positive result; - indicates a negative result.

[0057] Identification of 16S rRNA in strain XG-175:

[0058] DNA from strain XG-175 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 as follows: 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; 72℃ final extension for 10 min, and storage at 4℃. The PCR amplification 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.

[0059] BLAST comparison in NCBI revealed that the 16S rRNA gene sequence of strain XG-175 had a 99% similarity to that of Priestiamegaterium. Figure 5 As shown in the phylogenetic tree, strain XG-175 and Priestia megaterium (ATCC-14581) are both located in the smallest branch and are closely related in evolution. Based on the comprehensive physiological and biochemical indicators, strain XG-175 is identified as Priestia megaterium.

[0060] Example 2

[0061] The alkali-tolerant synergistic fermentation medium for Priestia megaterium XG-175 consists of: 18.0 g / L glucose, 22.0 g / L defatted soybean meal, 10.0 g / L sodium carboxymethyl cellulose, 6.0 g / L yeast extract, 5.0 g / L ammonium sulfate, 3.0 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, 0.3 g / L calcium chloride, 0.009 g / L ferrous sulfate, 2.5 g / L humic acid, 2.0 g / L urea, and deionized water.

[0062] The steps for culturing Priestia megaterium XG-175 using the above culture medium are as follows:

[0063] pH adjustment: Adjust to 8.5-9.0 using 1mol / L NaOH solution. After sterilization, the pH stabilizes at 8.3-8.8, requiring no secondary adjustment, which is suitable for the alkali-tolerant characteristics of the strain.

[0064] Sterilization conditions: autoclave at 121℃ for 20 minutes, cool to below 30℃ for aseptic inoculation (avoid high temperature to prevent damage to the activity of CMC-Na and soybean meal powder).

[0065] Inoculation rate: Inoculate Priestia megaterium XG-175 seed culture (OD) at 2% (v / v). 600 =0.8-1.0), shortening the strain's adaptation period;

[0066] Fermentation control: temperature 28℃, shaking speed 180rpm, fermentation cycle 48h;

[0067] Endpoint indicator: viable bacteria count ≥ 1.0 × 10⁻⁶ 9 CFU / mL, cellulase activity ≥130U / mL, protease activity ≥150U / mL, siderophore SU value ≥75%, organophosphorus lyase activity ≥90U / mL.

[0068] The comparison results of the core parameters of ordinary LB medium and the alkali-tolerant synergistic fermentation medium of the present invention are shown in Table 3.

[0069] Table 3 Comparison of core parameters between ordinary LB medium and the alkali-tolerant synergistic fermentation medium of this invention

[0070]

[0071] Example 3

[0072] Seed culture preparation: The cryopreserved strain Priestia megaterium XG-175 was inoculated into LB liquid 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;

[0073] Preparation of solid inoculum: Inoculate the seed culture at a rate of 1%-3% into an alkali-tolerant synergistic fermentation medium, and culture at 28℃ and 180 rpm for 48 hours until the viable count of Priestia megaterium XG-175 in the fermentation broth is ≥1.0 × 10⁻⁶. 9 CFU / mL; The Priestia megaterium XG-175 fermentation broth was mixed with sterile peat moss and well-rotted sawdust at a volume ratio of 1:4:1, and vacuum dried to a moisture content ≤15% to obtain a solid inoculum. The viable count of Priestia megaterium XG-175 was ≥1.0×10⁻⁶. 8 CFU / g;

[0074] Application in green spaces: Before tilling the soil in green spaces, evenly spread the solid microbial agent at a rate of 5-8 kg / mu on the soil surface, and then carry out shallow tilling (tillage depth 5-10 cm) to fully mix the solid microbial agent with the surface soil; for areas with accumulated dead branches and fallen leaves, a thin layer of soil (1-2 cm) can be covered after the microbial agent is spread to promote the contact between the Priestia megaterium XG-175 strain and organic pollutants;

[0075] Maintenance: After application, maintain soil moisture content at 20%-30%, no additional soil pH adjustment is required (suitable for pH≤11.0); apply solid microbial agent every 20-30 days (replenishment amount is 40% of the first application). The Priestia megaterium XG-175 strain colonizes the soil surface, degrades dead branches and fallen leaves through cellulase, and simultaneously degrades other pollutants through its functions of decomposing organic phosphorus and protein, producing iron carriers to enhance synergistic remediation efficiency.

[0076] The comparison results of indicators before and after treatment of organic polluted soil in urban green space using the method of this embodiment are shown in Table 4; where n is the number of samples tested, n=5, and the data in the table are the average values ​​of the measurements; treatment time: continuous operation for 45 days.

[0077] The method described in this embodiment is applicable to scenarios where the surface soil of urban green spaces is mainly polluted by fallen leaves and cellulose organic matter. It is suitable for large-scale pollution remediation of parks and residential green spaces, and can rapidly degrade surface organic pollutants.

[0078] Table 4 Comparison of indicators before and after solid microbial agent treatment of organically polluted soil in urban green spaces

[0079]

[0080] Example 4

[0081] Seed culture preparation: Cold-preserved Priestia megaterium XG-175 was inoculated into an alkali-tolerant functional synergistic 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;

[0082] Preparation of liquid inoculum: Inoculate the seed culture at a rate of 1%-3% into an alkali-tolerant synergistic fermentation medium, and culture at 28℃ and 180 rpm for 48 hours until the viable count of Priestia megaterium XG-175 in the fermentation broth is ≥1.0 × 10⁻⁶. 9 CFU / mL yields a liquid bacterial agent;

[0083] Dilution and application of microbial agent: Dilute the liquid microbial agent with water at a ratio of 1:500-1:800 to prepare a root irrigation solution; for trees and shrubs, apply by hole application, digging 3-5 holes 10-15cm deep around the plant roots, pouring 500-800mL of diluted microbial solution into each hole, and then covering with soil; for herbaceous plants, spray the diluted microbial solution evenly with a sprayer at a rate of 30-50L / acre;

[0084] Synergistic growth promotion: The strain colonizes around the plant roots, degrades organic phosphorus, protein and cellulose organic matter in the rhizosphere soil, and reduces the toxicity of pollutants to plants; the iron carrier can chelate soil iron and promote the absorption of nutrients by plants, achieving the dual effect of "pollution remediation + plant growth promotion"; the root irrigation solution is replenished every 25 days to ensure the activity of the rhizosphere strain.

[0085] The comparison results of indicators before and after the restoration of urban green space soil 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 60 days.

[0086] The method described in this embodiment is applicable to green spaces with severe local organic pollution, such as urban road green belts and landscape shrub areas. It is suitable for scenarios where organic phosphorus / protein accumulates in the middle layer of the soil, accurately targets and repairs polluted soil around the roots, and promotes the growth of green plants.

[0087] Table 5 Comparison of indicators before and after rhizosphere application of liquid bacterial agent for the remediation of urban green space soil (n=5, treatment time: continuous operation for 60 days)

[0088]

Claims

1. A bacterial strain for the remediation of organic pollution in urban green space soil, characterized in that, The strain is Priestiamegaterium XG-175, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38209.

2. The application of the strain described in claim 1 in the remediation of organic pollution in urban green space soil.

3. A microbial agent for the remediation of organic pollution in urban green space soil, characterized in that, The microbial agent includes Priestiamegaterium XG-175.

4. The microbial agent according to claim 3, characterized in that, The microbial agent can be a liquid or solid microbial agent.

5. An alkali-tolerant synergistic fermentation medium for Priestia megaterium XG-175, characterized in that, The culture medium comprises 18.0 g / L glucose, 22.0 g / L defatted soybean meal, 10.0 g / L sodium carboxymethyl cellulose, 6.0 g / L yeast extract, 5.0 g / L ammonium sulfate, 3.0 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, 0.3 g / L calcium chloride, 0.009 g / L ferrous sulfate, 2.5 g / L humic acid, and 2.0 g / L urea.

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