A method for water remediation by manganese-oxidizing bacteria- auxin synergistic in-situ induction of manganese spot on root surface of emergent plants

Abstract

This invention relates to the field of aquatic ecological restoration technology, and discloses a method for the in-situ induction of manganese spots on the root surface of emergent plants by synergistic induction of manganese-oxidizing bacteria and auxin. The method includes obtaining a suspension of manganese-oxidizing bacteria; mixing the manganese-oxidizing bacteria suspension, auxin, divalent manganese source, and basic plant nutrient solution to construct a synergistic induction solution; pretreating the roots of emergent plants in the synergistic induction solution; and transplanting the emergent plants loaded with biogenic manganese spots into the target polluted water body for adsorption and oxidative decomposition of the target pollutants. The auxin in the synergistic induction solution regulates the release of root exudates by emergent plants, promoting the colonization of manganese-oxidizing bacteria in the rhizosphere; the colonized manganese-oxidizing bacteria continuously oxidize divalent manganese ions, which are deposited on the root surface and root cortex of emergent plants to form high-valence biogenic manganese spots. This invention shortens the manganese spot formation cycle, utilizes the synergistic degradation of water pollutants by biogenic manganese spots and extracellular reactive oxygen species, and achieves in-situ recycling and regeneration of biogenic manganese spots in the restoration system.

Description

Technical Field

[0001] This invention relates to the field of aquatic ecological restoration technology, specifically a method for the in-situ induction of manganese spots on the root surface of emergent plants by the synergistic action of manganese-oxidizing bacteria and auxin. Background Technology

[0002] Microbial-plant coupling technology is an important method in the field of aquatic ecological restoration. Manganese-oxidizing bacteria can attach to the root surface of aquatic plants in natural environments and induce the formation of biogenic manganese spots. Compared with manganese oxides formed through chemical processes, biogenic manganese oxides have weaker crystallinity, higher manganese valence states, more cavities, and a larger specific surface area, exhibiting stronger reactivity. Biogenic manganese oxides generated by in-situ coupled functional bacteria in the rhizosphere possess adsorption and oxidation properties for heavy metals, ammonia nitrogen, and organic pollutants, showing potential application in the purification of polluted water bodies.

[0003] Existing microbial-plant coupled remediation systems employ various approaches to enhance pollutant removal in practical applications. Some remediation methods involve surface carboxyl modification of granular biomass obtained from dried and pulverized aquatic plants to enhance the pollutant response. Others utilize fungi such as *Moses* to infect plant roots for the treatment of eutrophic water bodies. In water treatment systems involving phytomanganese spot induction, current technologies typically involve directly laying manganese dioxide-containing manganese ore substrates at the bottom of culture containers or in river purification devices, or introducing manganese oxides using artificially synthesized microbial carriers, inducing rhizosphere manganese spot formation through passive absorption by plant growth and physical contact with the roots.

[0004] Existing technologies for inducing manganese spots on the root surface of aquatic plants suffer from slow system initiation and low colonization efficiency. When establishing rhizosphere systems through natural generation or fungal infection, the time required for fungal culture and root infection often reaches tens of days, resulting in an excessively long initiation period for the overall remediation system. Furthermore, methods using direct application of manganese ore or artificial carriers to induce manganese spots rely on non-renewable solid mineral matrices, which are not only costly and resource-intensive, but also restrict the manganese spot induction process due to spatial and interface limitations. This leads to unstable bacterial colonization on the plant root surface, making it impossible to achieve rapid in-situ induction and uniform, stable loading of manganese spots on the root surface within a short period. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for the in-situ induction of manganese spots on the root surface of emergent plants by manganese-oxidizing bacteria and auxin synergy, thus solving the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for the in-situ induction of manganese spots on the root surface of emergent plants by synergistic effects of manganese-oxidizing bacteria and auxin, comprising the following steps: Obtain a manganese-oxidizing functional bacterial suspension that reaches a preset cell concentration, wherein the microorganisms in the manganese-oxidizing functional bacterial suspension are manganese-oxidizing bacteria; The manganese-oxidizing functional bacterial suspension, plant hormone, divalent manganese source and plant basic nutrient solution are mixed to construct a synergistic induction solution, wherein the plant hormone is auxin; The root system of the emergent plant to be treated is placed in the synergistic induction solution for pretreatment, and in-situ induced biogenic manganese spots are obtained on the root surface of the emergent plant. Emergent plants loaded with the aforementioned biogenic manganese spots are transplanted into the target polluted water body, where the in-situ biogenic manganese spots in the rhizosphere of the emergent plants adsorb and oxidize the target pollutants in the target polluted water body.

[0007] Furthermore, the manganese-oxidizing bacteria is Pseudomonas putida MnB1; the plant basal nutrient solution is Hoagland nutrient solution; and the divalent manganese source is manganese sulfate monohydrate.

[0008] Furthermore, when preparing the synergistic induction solution, the concentration range of manganese sulfate monohydrate is set from 0 mg / L to 20 mg / L, and the concentration range of auxin is set from 0 mg / L to 1.0 mg / L; the initial pH of the synergistic induction solution is adjusted to 7.0, and the temperature is set to 25°C; during the preparation process, fresh induction solution is used to suspend the bacterial cells, and the cell concentration of *Pseudomonas putida* MnB1 is adjusted to achieve an absorbance OD of the bacterial solution. 600 The preset values ​​are achieved to meet the linear fitting requirements of cell concentration.

[0009] Furthermore, the emergent plant is one of the following: yellow iris, reed, or cattail. In the pretreatment, the surface of the synergistic induction solution is controlled to completely cover the root system of the emergent plant, and the synergistic induction solution is stirred at a set frequency.

[0010] Furthermore, after the pretreatment, a flocculent microbial film forms on the root surface of the emergent plant, and nano-manganese oxide is deposited within the cells and intercellular spaces of the outer cortex of the emergent plant root. The crystal structure of the deposited biogenic manganese spots corresponds to the MnO2 and Mn2O3 standard cards, and the biogenic manganese spots are mainly composed of trivalent and tetravalent manganese.

[0011] The innovative principle of this invention's water remediation method lies in constructing a synergistic microecological system of plant hormones and manganese-oxidizing bacteria within the rhizosphere environment of emergent plants. The added auxin regulates the physiological metabolism and stress resistance of emergent plants, prompting them to release root exudates. These exudates serve as a natural carbon source, maintaining and promoting the stable colonization of manganese-oxidizing bacteria on the root surface of emergent plants. Under conditions of physical agitation maintaining material exchange, the colonized manganese-oxidizing bacteria continuously oxidize divalent manganese ions in the environment, causing nano-sized high-valence manganese oxide to deposit on the root surface of emergent plants and penetrate to the outer layer of the root cortex, forming firmly bonded bio-manganese spots in situ. This solves the problems of low bacterial colonization rate and long manganese spot formation cycle in traditional systems.

[0012] During the transplantation remediation phase, the trivalent and tetravalent manganese contained in the biogenic manganese patches directly provide oxidation reaction sites for the degradation of pollutants in the water. Simultaneously, the surface of emergent plant roots, in synergy with bacteria, enriches manganese oxidation genes containing multi-copper oxidases and produces extracellular reactive oxygen species. These extracellular reactive oxygen species synergistically catalyze oxidation reactions with the biogenic manganese patches, achieving bond-breaking degradation and removal of target pollutants such as ammonia nitrogen, 4-chlorophenol, and sulfamethoxazole from eutrophic water bodies and water bodies containing recalcitrant organic pollutants. During the pollutant treatment process, the manganese ions reduced through oxidation are re-oxidized from the water and replenished as high-valence manganese oxides under the biocatalytic action of manganese-oxidizing bacteria continuously colonizing the surface of emergent plant roots. This process achieves in-situ recycling and regeneration of biogenic manganese patches in the remediation system, maintaining the long-term operation of the water remediation.

[0013] This invention provides a method for the in-situ induction of manganese spots on the root surface of emergent plants by synergistic effects of manganese-oxidizing bacteria and auxin. It has the following beneficial effects: 1. This invention shortens the formation cycle of biogenic manganese spots on the root surface of emergent plants by constructing a microecological induction system synergistically involving manganese-oxidizing bacteria and plant auxins. Under divalent manganese source conditions, auxins regulate the release of secretions from plant roots, providing a carbon source for manganese-oxidizing bacteria and promoting their colonization in the rhizosphere. The colonized manganese-oxidizing bacteria continuously oxidize manganese ions in the aquatic environment, causing manganese oxide to be deposited on the root surface of emergent plants and in the intracellular and intercellular spaces of the outer layer of the root cortex. This mechanism overcomes the shortcomings of slow initiation of conventional fungal infection and high cost of artificial substrate laying, enabling the in-situ construction of a water remediation system.

[0014] 2. This invention enhances the purification capacity of a single plant system for recalcitrant organic pollutants and eutrophic water bodies by utilizing the synergistic effect of biogenic manganese deposits on the root surface and extracellular reactive oxygen species mediated by multi-copper oxidases. The deposited biogenic manganese deposits contain trivalent and tetravalent manganese phases, providing oxidation reaction sites for pollutant degradation. High-valence manganese oxides, combined with environmentally activated reactive oxygen species, adsorb and oxidize chlorinated organic pollutants, recalcitrant antibiotics, and ammonia nitrogen, thereby strengthening the pollution reduction efficiency of the plant system.

[0015] 3. This invention achieves in-situ recycling and regeneration of biogenic manganese spots through the biocatalytic action of manganese-oxidizing bacteria, simultaneously enhancing the stress resistance and growth capacity of emergent plants. During the water treatment process, low-valence manganese ions, which participate in the pollution reduction reaction and are reduced, are re-oxidized into high-valence manganese oxides by functional bacteria colonizing the root surface and deposited. This cyclic replenishment process maintains the stable operation of the remediation system, avoiding the defects of single-use failure of conventional solid mineral matrices. The deposition of biogenic manganese spots promotes the thickening of the intercellular layer of plant cells, and the entire process does not require the laying of non-recyclable substrates, avoiding the risk of secondary environmental pollution. Attached Figure Description

[0016] Figure 1 Analysis of the manganese oxidation characteristics of MnB1 in Examples 1 and 2 of this invention: (a) Colony morphology during enrichment in PYCM medium; (b) Identification of biogenic manganese oxide with LBB chromogenic reagent (blue positive); (c) Stereoscopic micrograph of biogenic manganese oxide; (d) XRD phase analysis of biogenic manganese oxide.

[0017] Figure 2 The samples are the treatment group (MnB1+IAA+Mn²⁺) and the natural plant control group in the XPS valence state analysis of manganese element in the root biofilm of the present invention in Examples 1 and 4 of the present invention.

[0018] Figure 3 These are the removal effect diagrams of 4-chlorophenol by the method of the present invention in Examples 1, 4, and 5 of the present invention.

[0019] Figure 4 The method of the present invention in Comparative Examples 1 and 2 demonstrates the removal efficiency and cyclic stability of sulfamethoxazole by the method of the present invention.

[0020] Figure 5 This refers to the changes in root surface manganese content and bacterial colonization with IAA concentration during the pretreatment stage in Example 3 of the present invention: root surface manganese content / corresponding bacterial colonization at different IAA concentrations.

[0021] Figure 6 The image shows a comparison of root cell morphological changes in the experimental plants in Example 1 of this invention; where (a) is the Pseudomonas genus functional bacteria coupling group; and (b) is the sterile plant group. Detailed Implementation

[0022] The technical solutions in 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. Example

[0023] Please see the appendix Figure 1 - Appendix Figure 6 This invention provides a method for the in-situ induction of manganese spots on the root surface of emergent plants by manganese-oxidizing bacteria and auxin synergy, comprising the following steps: S100. Preparation and activation of manganese-oxidizing functional bacterial suspension. A specific genus of manganese-oxidizing bacteria was selected for inoculation and aerobic enrichment culture to obtain a manganese-oxidizing functional bacterial suspension reaching a preset cell concentration. In this invention, the specific manganese-oxidizing bacteria is *Pseudomonas putida* MnB1. The preparation process of the manganese-oxidizing functional bacterial suspension includes the activation of frozen *Pseudomonas putida* MnB1, aerobic enrichment culture, and resuspending the cells in fresh solution to adjust the cell concentration. For the preparation and sterilization of the conventional component culture media involved in the aerobic enrichment culture step, those skilled in the art can refer to the conventional microbiology laboratory manual for preparation, which is well-known in the field and will not be elaborated here.

[0024] S200. Preparation of the synergistic induction solution. The manganese-oxidizing functional bacterial suspension prepared in step S100, plant hormones, divalent manganese source, and basic plant nutrient solution are mixed to construct the synergistic induction solution. In the constructed synergistic induction solution, the plant hormone is specifically indole-3-acetic acid (IAA); the divalent manganese source is specifically manganese sulfate monohydrate; and the basic plant nutrient solution is Hoagland solution. The synergistic induction solution, by controlling the added concentrations of manganese-oxidizing bacteria, manganese sulfate monohydrate, and auxin, allows manganese-oxidizing bacteria to grow and reproduce on the roots of emergent plants, while avoiding the potential phytotoxic effects of excessive manganese ions. Auxin is used to regulate the mutually beneficial symbiotic process between the root exudates of emergent plants and manganese-oxidizing bacteria, promoting the stable loading of manganese spots on the root surface.

[0025] S300. In-situ pretreatment of emergent plant rhizosphere and induction of biogenic manganese spots. The root system of the emergent plant to be treated is immersed in the synergistic induction solution constructed in step S200 to perform in-situ pretreatment. The emergent plants applicable to this invention include any one of yellow iris, reed, or cattail. During the in-situ pretreatment, physical agitation at a set frequency is used to promote full contact between microorganisms and the roots of aquatic plants; Pseudomonas putida MnB1 attaches to and colonizes on the root surface of emergent plants, forming a flocculent microbial film, while oxidizing divalent manganese ions in the synergistic induction solution. Nano-manganese oxide is deposited in the intracellular and intercellular spaces of the outer layer of the inner cortex of the emergent plant root, forming biogenic manganese spots in situ on the root surface of the emergent plant. The crystal structure of the deposited biogenic manganese spots mainly includes MnO2 and Mn2O3 phases.

[0026] S400: In-situ water remediation with emergent plants bearing biogenic manganese spots. Emergent plants pretreated in step S300 and bearing biogenic manganese spots are transplanted into the target polluted water body. The target polluted water body includes eutrophic water or water containing recalcitrant organic matter, and the target pollutants include 4-chlorophenol, sulfamethoxazole, or ammonia nitrogen. Emergent plants bearing biogenic manganese spots utilize extracellular reactive oxygen species generated by the enriched expression of copper oxidase genes on their root surfaces, combining with the oxidation reaction sites provided by the biogenic manganese spots to adsorb and oxidize the target pollutants in the target polluted water body. During the water treatment process, the biogenic manganese spots consumed on the root surface are continuously replenished with manganese in the water body by the colonized Pseudomonas putida MnB1, achieving in-situ regeneration of biogenic manganese spots and remediation of water pollution.

[0027] The preparation and activation process of the manganese-oxidizing functional bacterial suspension in step S100 specifically includes the following sub-steps: S101. Preparation of Peptone Yeast Extract Manganese Medium (PYCM). The specific component concentrations of PYCM are as follows: 0.8 g / L peptone, 0.2 g / L yeast extract, 0.1 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.2 g / L sodium nitrate, 0.1 g / L calcium chloride, 0.1 g / L ammonium chloride, 1.0 g / L ferric ammonium citrate, 0.2 g / L manganese sulfate, and 0.1 g / L ammonium carbonate. After mixing all components, adjust the pH of the PYCM to 7.0.

[0028] S102, activated lyophilized Pseudomonas putida MnB1. The strain used was purchased from the Shanghai Biotechnology Preservation Center, with accession number SHBCC D25196 (=ATCC 23483). The lyophilized powder was activated using NA solid medium, the composition of which included 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, and 15.0 g / L agar.

[0029] S103. Activated Pseudomonas putida MnB1 was inoculated into peptone yeast extract manganese medium for aerobic enrichment culture. Pseudomonas putida MnB1 was inoculated into peptone yeast extract manganese medium at a transfer rate of 3% (v / v). The culture was set at 30℃ and aerobic enrichment culture was carried out for 2 days with horizontal shaking at 150 rpm to obtain Pseudomonas putida MnB1 bacterial suspension.

[0030] The preparation of the synergistic induction solution in step S200 specifically includes the following sub-steps: S201. Adjust the cell concentration of *Pseudomonas putida* MnB1. Place the *Pseudomonas putida* MnB1 bacterial suspension obtained in step S103 into a centrifuge and centrifuge at 4000 rpm for 10 minutes. After centrifugation, discard the supernatant and resuspend the cells in an equal volume of fresh co-induction medium. Measure the absorbance (OD) of the bacterial suspension. 600 Adjusted to 0.29. During the resuspending of the bacterial cells, the cell number and absorbance OD of Pseudomonas putida MnB1 were compared. 600 It conforms to a linear fit, and the specific calculation formula is: y = 3.35 × 10 7 x.

[0031] In the calculation formula, This indicates the number of cells per unit volume of bacterial culture, expressed in cells / mL. Indicates absorbance OD 600 Measured values; correlation coefficient of the calculation formula According to the calculation formula, absorbance OD 600 A value of 0.29 corresponds to a cell number of Pseudomonas putida MnB1 reaching [a certain value]. The level of cells / mL.

[0032] S202. Add plant basal nutrient solution and divalent manganese source to the synergistic induction solution. Add 10% (v / v) Hoagland nutrient solution to the synergistic induction solution to provide the basic elements required for the growth of aquatic plants. Add manganese sulfate monohydrate to the synergistic induction solution as a reaction substrate for the formation of biogenic manganese spots. The concentration range of manganese sulfate monohydrate is set from 0 mg / L to 20 mg / L. To avoid the phytotoxic effects of excessive manganese ions, the preferred concentration of manganese sulfate monohydrate is set at 10 mg / L.

[0033] S203. Add plant hormones to the synergistic induction solution. Auxin IAA is added to the synergistic induction solution as a plant hormone signaling substance. The concentration range of auxin IAA is set from 0 mg / L to 1.0 mg / L. The preferred concentration of auxin IAA is set to 0.3 mg / L. After mixing, the initial pH of the synergistic induction solution is set to 7.0, and the temperature is set to 25℃. The added auxin IAA is used to regulate the stress resistance, growth metabolism, and expression of pollutant-degrading enzymes of emergent plants. Under the regulation of auxin IAA, emergent plants release root exudates, which serve as a carbon source to enrich and maintain the stable growth of *Pseudomonas putida* MnB1 on the root surface of emergent plants. Auxin IAA regulates the mutualistic symbiotic process between the root exudates of emergent plants and *Pseudomonas putida* MnB1, promoting the stable loading of *Pseudomonas putida* MnB1 on the root surface of emergent plants.

[0034] After preparing the synergistic induction solution in step S200, the solution is used to perform step S300: in-situ rhizosphere pretreatment and induction of biogenic manganese spots in emergent plants. Step S300 specifically includes the following sub-steps: S301. Select emergent plants to be treated. Specifically, the selected emergent plants are one of the following: yellow iris, reed, or cattail. Taking yellow iris as an example, select seedlings of the aquatic plant yellow iris with a leaf length of 30±5 cm and an average root length of 20±5 cm for later use.

[0035] S302. Place the roots of the emergent plants selected in step S301 into the synergistic induction solution prepared in step S200 for pretreatment. During pretreatment, ensure that the surface of the synergistic induction solution completely covers the roots of the emergent plants, set the distribution density of the emergent plants to approximately 70%, and set the temperature of the synergistic induction solution to 25°C.

[0036] S303. The pretreatment time is set to 10 days. During the 10-day pretreatment, the synergistic induction solution is stirred every 12 hours to promote full contact between the microorganisms in the synergistic induction solution and the roots of emergent plants.

[0037] S304. After pretreatment, in-situ induced biogenic manganese spots are obtained on the surface of emergent plant roots. A visible flocculent microbial film forms on the surface of emergent plant roots. During the pretreatment process, nano-manganese oxide is deposited on the surface of emergent plant roots and in the intracellular and intercellular spaces of the outer layer of the root cortex. The intercellular layer of emergent plant roots is thickened, and manganese is enriched per unit root surface area. The crystal structure deposited on the surface of emergent plant roots corresponds to the standard cards of MnO2 and Mn2O3, and the content of extracellular reactive oxygen species (ROS) in the root biofilm is increased.

[0038] S305. Taking *Iris tectorum* as an example, after 10 days of pretreatment, the colonization efficiency of *Pseudomonas putida* MnB1 on the root surface of *Iris tectorum* reached [percentage missing]. cells / cm 2 The manganese enrichment content per unit root surface area of ​​*Iris tectorum* was 5.62 ± 0.04 mg / cm³. 2 The sum of the two phases, MnO2 and Mn2O3, accounts for 42.20% of the total manganese content in the biogenic manganese spots. The emergent plants of yellow iris exhibit enhanced resistance and accelerated growth, with the actual plant height increasing by 21% compared to the initial plant height.

[0039] After completing step S300, proceed to step S400 for in-situ remediation of emergent plants loaded with biogenic manganese spots. Step S400 specifically includes the following sub-steps: S401. Transplant the emergent plants, pretreated in step S300 and loaded with biogenic manganese spots, into the target polluted water body. The target polluted water body includes eutrophic water bodies and polluted water bodies containing recalcitrant organic pollutants. The target pollutants include ammonia nitrogen, 4-chlorophenol, or sulfamethoxazole.

[0040] S402. In-situ biogenic manganese spots in the rhizosphere of emergent plants adsorb and oxidize target pollutants in polluted water bodies. The biogenic manganese spots are predominantly composed of trivalent and tetravalent manganese, which provide oxidation reaction sites for the degradation of target pollutants. The rhizosphere of emergent plants containing biogenic manganese spots synergistically treats target pollutants in the water body through extracellular reactive oxygen species and biogenic manganese spots. Taking *Iris tectorum* as an example, under a static remediation state at 25℃, after a 72-hour treatment cycle, a single batch of *Iris tectorum* loaded with biogenic manganese spots achieved a 92.6% removal rate for ammonia nitrogen at an initial concentration of 10 mg / L and an 88.4% removal rate for 4-chlorophenol at an initial concentration of 1 mg / L. For *Phragmites australis* and *Typha orientalis*, the removal rates for 1 mg / L 4-chlorophenol polluted water after 72 hours of treatment were 81.2% and 83.5%, respectively.

[0041] S403. In-situ recycling and regeneration of biogenic manganese spots during the treatment of polluted water. During the treatment of the target pollutant, the biogenic manganese spots are consumed by the reaction. The consumed biogenic manganese spots, under the action of *Pseudomonas putida* MnB1 colonized on the root surface of emergent plants, continuously replenish the manganese in the target polluted water as biogenic manganese spots, maintaining the remediation of the target polluted water. Taking a target polluted water containing 1 mg / L sulfamethoxazole as an example, the same batch of treated *Iris tectorum* was continuously transferred into three batches of freshly prepared target polluted water containing sulfamethoxazole. Each batch was run for 72 hours, without supplementation of *Pseudomonas putida* MnB1 bacterial solution, auxin IAA, or manganese sulfate monohydrate. In continuous operation tests, the removal rate of sulfamethoxazole by the first batch of yellow iris was 86.7%. After three consecutive batches, the removal rate of sulfamethoxazole by yellow iris stabilized at 82.1%, achieving long-term stable operation of bio-manganese spots.

[0042] To further support and verify the water remediation method described above with specific experimental data, specific embodiments are provided below.

[0043] Example 1: Optimal process combination for remediation of water bodies containing 4-chlorophenol contaminants Yellow iris seedlings with leaf length of 30±5 cm and root length of 20±5 cm were selected for use. A manganese spot induction solution was prepared, containing absorbance OD... 600 The induction solution consisted of 0.29 g / L Pseudomonas putida MnB1 bacterial culture, 10% Hoagland nutrient solution, 10 mg / L manganese sulfate monohydrate, and 0.3 mg / L IAA auxin. Sterile pure water was used to bring the solution to volume, the initial pH was adjusted to 7.0, and the temperature was set at 25°C.

[0044] The roots of young yellow iris seedlings were completely submerged in the induction solution, with a leaf coverage rate controlled at 70%. The pretreatment period was set at 10 days, and the induction solution was stirred every 12 hours. After pretreatment, uniform brown manganese spots formed on the root surface of the yellow iris seedlings. Tests showed that the manganese content per unit root surface area was 5.62 ± 0.04 mg / cm². 2 The relative fluorescence intensity of extracellular reactive oxygen species was 1500±200 RFU, and the plant height increased by 21% compared with the initial value.

[0045] Manganese-laden *Iris tectorum* was introduced into simulated contaminated water. The simulated contaminated water contained an initial concentration of 10 mg / L ammonia nitrogen and an initial concentration of 1 mg / L 4-chlorophenol. After 72 hours of remediation at 25°C, the ammonia nitrogen removal rate was measured to be 92.6%, and the 4-chlorophenol removal rate was 88.4%. For the detection of ammonia nitrogen and 4-chlorophenol concentrations in the water, those skilled in the art can refer to conventional standard methods for water quality analysis. The relevant water quality testing procedures are well-known techniques in the field and will not be described in detail in this specification.

[0046] Example 2: Effect of manganese sulfate monohydrate concentration gradient on manganese spot characteristics in the system The concentration of manganese sulfate monohydrate in the induction solution was varied to create five concentration gradients: 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L. All other process parameters remained the same as in Example 1.

[0047] After pretreatment, the manganese content on the root surface of *Iris tectorum* seedlings was tested. When the manganese sulfate monohydrate concentration was 0 mg / L, no obvious manganese spots were observed on the root surface, and the manganese content was 0.12 mg / cm³. 2 When the concentration of manganese sulfate monohydrate is 5 mg / L, the manganese content on the root surface is 2.35 mg / cm³. 2 The manganese spots were sparsely distributed. When the concentration of manganese sulfate monohydrate was 10 mg / L, the manganese content on the root surface reached a peak of 5.62 mg / cm³. 2 The manganese spots were uniform and dense. In the high concentration groups of manganese sulfate monohydrate (15 mg / L and 20 mg / L), the leaf tips of *Iris tectorum* turned yellow, and the manganese content on the root surface showed no upward trend, indicating manganese ion toxicity. Experimental data verified that the optimal concentration of manganese sulfate monohydrate in this invention, while avoiding manganese ion toxicity, is 10 mg / L.

[0048] Example 3: Effect of auxin IAA concentration gradient on manganese spot characteristics in the system The concentration of auxin IAA in the induction solution was varied, with five concentration gradients set at 0 mg / L, 0.1 mg / L, 0.3 mg / L, 0.5 mg / L, and 1.0 mg / L. All other process parameters remained the same as in Example 1.

[0049] Test data showed that in the hormone-free group with an IAA concentration of 0 mg / L, the root surface manganese content was 3.48 mg / cm². 2 The bacterial colony size was 5.2 × 10⁻⁶. 6 cells / cm 2The relative fluorescence intensity of extracellular reactive oxygen species was 620±80 RFU, and the plant height increased by 8%. In the group with an IAA concentration of 0.3 mg / L, all indicators reached their corresponding peak values, with the root surface manganese content at 5.62 mg / cm³. 2 The bacterial colony size was 1.12 × 10⁻⁶. 7 cells / cm 2 Plant height increased by 21%. When the concentration of auxin IAA was greater than or equal to 0.5 mg / L, all indicators tended to stabilize, with no significant increase in efficacy. Experimental data demonstrated that auxin IAA enhanced bacterial colonization and manganese deposit deposition, and verified that the optimal concentration of auxin IAA was 0.3 mg / L.

[0050] Example 4: Manganese spot characteristics in a system without manganese-oxidizing bacteria Prepare the induction solution. Do not add *Pseudomonas putida* MnB1 bacterial culture to the induction solution; only retain 10% (v / v) Hoagland nutrient solution, 10 mg / L manganese sulfate monohydrate, and 0.3 mg / L auxin IAA. Adjust the initial pH of the induction solution to 7.0 and set the temperature to 25°C.

[0051] The roots of young yellow iris seedlings were pretreated by immersing them in an induction solution. The pretreatment conditions were consistent with those in Example 1. After 10 days of pretreatment, no visible manganese spots were observed on the root surface of the young yellow iris seedlings. The manganese content on the root surface of the young yellow iris seedlings was measured to be 0.18 mg / cm³. 2 .

[0052] Pretreated yellow iris was transferred into simulated contaminated water containing an initial concentration of 1 mg / L of 4-chlorophenol for 72 hours for remediation. Test results showed that the removal rate of 4-chlorophenol in the simulated contaminated water was 12.3%.

[0053] Experimental results demonstrate that Pseudomonas putida MnB1 is a necessary condition for the formation of manganese spots on the root surface of emergent plants and for the remediation of water pollution; non-functional bacteria cannot achieve manganese spot construction and pollution control.

[0054] Example 5: Manganese spot characteristics of the system under different aquatic plant conditions The yellow iris seedlings in Example 1 were replaced with emergent plants reed and cattail. The components of the induction solution and the pretreatment process parameters remained unchanged from those in Example 1.

[0055] The roots of reed seedlings and cattail seedlings were pretreated by immersing them in an induction solution for 10 days. After 10 days of pretreatment, manganese spots formed on the root surface of the reed seedlings, and the manganese content on the root surface of the reed seedlings was measured to be 4.91 mg / cm³.2 Manganese spots formed on the root surface of cattail seedlings, and the manganese content on the root surface of the cattail seedlings was found to be 5.03 mg / cm³. 2 .

[0056] Manganese-laden reeds and cattails were respectively introduced into simulated polluted water containing 4-chlorophenol at an initial concentration of 1 mg / L. After 72 hours of operation, the test results showed that the removal rate of 4-chlorophenol by manganese-laden reeds was 81.2%, and the removal rate of 4-chlorophenol by manganese-laden cattails was 83.5%.

[0057] Experimental data demonstrate that water remediation methods are not limited to yellow iris; they can be adapted to emergent plants including reeds and cattails.

[0058] Comparative Example 1: Hormone-synergistic enhancement of manganese spot phytoremediation of recalcitrant organic matter sulfamethoxazole Yellow iris loaded with biogenic manganese spots was prepared according to the scheme of Example 1. Yellow iris from the same batch was consecutively transferred into three batches of freshly prepared simulated contaminated water. The simulated contaminated water contained sulfamethoxazole, a recalcitrant organic compound, at an initial concentration of 1 mg / L. The treatment time for each batch of simulated contaminated water was 72 hours. Throughout the entire treatment process for the three consecutive batches, no Pseudomonas putida MnB1, no auxin IAA, and no manganese sulfate monohydrate were supplemented.

[0059] Tests showed that in the first batch of 72-hour treatment, the removal rate of sulfamethoxazole by *Iris tectorum* bearing manganese spots was 86.7%, while the removal rate by the untreated control group was only 23.4%. After three consecutive batches, the removal rate of sulfamethoxazole by *Iris tectorum* bearing manganese spots remained stable at 82.1%, without any decline. These data demonstrate that the system provided by this invention possesses in-situ regeneration capabilities, achieving stable cyclic operation and solving the problem of one-time failure in traditional remediation systems.

[0060] Comparative Example 2: Manganese Mound Plant System Based on Manganese Ore Laying A control group with a traditional manganese ore substrate was set up. A 2 cm thick layer of manganese ore with a particle size of 2 to 4 mm and an MnO2 content greater than or equal to 35% was laid at the bottom of the culture container. Hogrange nutrient solution (10% by volume) was added and changed weekly. Yellow iris seedlings of the same size were transplanted, without the addition of Pseudomonas putida MnB1 or auxin IAA, and cultured conventionally for 60 days.

[0061] Comparative results showed that after 60 days of cultivation using the traditional manganese ore spreading method, the manganese content on the root surface of *Iris tectorum* was 3.52 mg / cm²; the removal rate after 72 hours of sulfamethoxazole treatment was 54.3%. In contrast, Example 1 of this invention achieved a root surface manganese enrichment of 5.62 mg / cm² after only 10 days of pretreatment, with a removal rate of 86.7%. Traditional techniques suffer from problems such as high manganese ore consumption, high cost, inability to recycle, and easy accumulation leading to secondary pollution. The method described above requires no solid matrix, is simple to operate, and avoids the risk of secondary pollution.

[0062] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the in-situ induction of manganese spots on the root surface of emergent plants by synergistic effects of manganese-oxidizing bacteria and auxin, characterized in that, Includes the following steps: Step S100: Obtain a manganese-oxidizing functional bacterial suspension that reaches a preset cell concentration, wherein the microorganisms in the manganese-oxidizing functional bacterial suspension are manganese-oxidizing bacteria; Step S200: Mix the manganese-oxidizing functional bacterial suspension, plant hormone, divalent manganese source and plant basic nutrient solution to construct a synergistic induction solution, wherein the plant hormone is auxin; Step S300: The root system of the emergent plant to be treated is placed in the synergistic induction solution for pretreatment, and in-situ induced bio-manganese spots are obtained on the root surface of the emergent plant. Step S400: Transplant the emergent plant loaded with the biogenic manganese spots into the target polluted water body. The in-situ biogenic manganese spots in the rhizosphere of the emergent plant adsorb and oxidize the target pollutants in the target polluted water body.

2. The water body remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 1, characterized in that, In step S100, the manganese-oxidizing bacteria is Pseudomonasputida MnB1. In step S200, the plant basic nutrient solution is Hoagland nutrient solution, and the divalent manganese source is manganese sulfate monohydrate.

3. The water body remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 2, characterized in that, Step S100 includes: Preparation of manganese culture medium for peptone yeast extract; Obtain the purified and activated Pseudomonas putida MnB1; The Pseudomonas putida MnB1 strain was inoculated into the peptone yeast extract manganese medium for aerobic enrichment culture to obtain Pseudomonas putida MnB1 bacterial suspension.

4. The water body remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 3, characterized in that, Step S200 includes: After centrifuging the Pseudomonas putida MnB1 bacterial suspension, the cells were resuspended in freshly prepared synergistic induction medium to adjust the cell concentration of Pseudomonas putida MnB1. The absorbance OD of the bacterial suspension was then measured. 600 Adjust to the preset value; Add 10% by volume of the Hogland nutrient solution to the synergistic induction solution, and add manganese sulfate monohydrate at a concentration within a set range to the synergistic induction solution; Add the auxin to the synergistic induction solution at a set concentration range, adjust the initial pH of the synergistic induction solution, and set the temperature of the synergistic induction solution.

5. The water remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 4, characterized in that, In step S200, the concentration range of the added manganese sulfate monohydrate is 0 mg / L to 20 mg / L; the concentration range of the added auxin is 0 mg / L to 1.0 mg / L; the initial pH value is 7.0; and the temperature is 25°C.

6. The water body remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 1, characterized in that, Step S300 includes: Select the emergent plant to be treated, which is one of yellow iris, reed or cattail; The roots of the selected emergent plants are placed in the synergistic induction solution for pretreatment, and the liquid surface of the synergistic induction solution is controlled to completely cover the roots of the emergent plants, and the distribution density of the emergent plants is set. The pretreatment time is set to 10 days, and the synergistic induction liquid is stirred at a set frequency during the pretreatment. After the pretreatment, the biogenic manganese spots induced in situ are obtained on the root surface of the emergent plant.

7. The water body remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 6, characterized in that, During the process of obtaining the bio-manganese spots induced in situ, a flocculent microbial film forms on the root surface of the emergent plant; In the pretreatment, nano-manganese oxide is deposited on the surface of the emergent plant roots and in the cells and intercellular spaces of the outer layer of the root cortex, thereby thickening the intercellular layer of the emergent plant roots.

8. The water body remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 7, characterized in that, The crystal structure of the deposited biogenic manganese spots corresponds to the MnO2 and Mn2O3 standard cards; The presence of the biogenic manganese spots is mainly composed of trivalent and tetravalent manganese; The emergent plants exhibited enrichment of copper oxidase-type manganese oxidation genes on the root surface, leading to an increase in the content of extracellular reactive oxygen species in the root biofilm.

9. The water remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 1, characterized in that, In step S400, the target polluted water body includes eutrophic water body and polluted water body containing recalcitrant organic pollutants; the target pollutants include ammonia nitrogen, 4-chlorophenol or sulfamethoxazole.

10. The water body remediation method for manganese-oxidizing bacteria-auxin synergistic in situ induction of manganese spots on the root surface of emergent plants according to claim 9, characterized in that, In step S400, the rhizosphere of emergent plants loaded with the biogenic manganese spots synergistically treats the target pollutants in the target polluted water body by generating extracellular reactive oxygen species in the biogenic manganese spots. During the treatment of the target pollutant, the biogenic manganese spot is consumed by the reaction; The consumed bio-manganese spots, under the action of the manganese-oxidizing bacteria colonizing the root surface of the emergent plants, continuously replenish the manganese in the target polluted water body as bio-manganese spots, thereby realizing the in-situ regeneration of bio-manganese spots and the remediation of water pollution.

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