In-situ remediation method for arsenic pollution based on chara corrhosa-nitraria sibirica symbiotic system

Abstract

The application discloses an arsenic pollution in-situ remediation method based on a Nitzschia palea-Vallisneria natans symbiotic system and belongs to the technical field of pollution prevention and control. The remediation method takes Nitzschia palea as a functional algae species, and a stable attached symbiotic system is constructed with Vallisneria natans to realize in-situ remediation of arsenic pollution. In the application, Nitzschia palea is specifically attached to the surface of the rhizome of Vallisneria natans by virtue of a siliceous cell wall, and the absorption and enrichment of arsenic by Vallisneria natans are significantly strengthened through interface binding site amplification, rhizosphere arsenic form activation, physiological stress resistance protection and other effects; under the stress of 100 mu g / L arsenic, the arsenic enrichment stability of Vallisneria natans is improved by 15.29% compared to single Vallisneria natans, and the arsenic concentration in water can be efficiently reduced.

Description

Technical Field

[0001] This invention relates to the fields of pollution prevention and control, water environment pollution treatment and ecological restoration technology, and to an in-situ remediation method for arsenic pollution based on the symbiotic system of *Nyctaginella tamariscina* and *Vallisneria natans*. Specifically, it relates to a method for in-situ ecological restoration of water bodies with low concentrations of arsenic pollution based on the symbiotic system of *Nyctaginella tamariscina* and *Vallisneria natans*. Background Technology

[0002] Arsenic is a highly toxic metallic pollutant widely present in the aquatic environment. Long-term exposure to low doses can still cause irreversible damage to human health and aquatic ecosystems. Many lakes, reservoirs, drinking water sources, and agricultural drainage systems contain low concentrations of arsenic pollution. These water bodies are characterized by low pollution concentrations, large water areas, and high ecological sensitivity, making physical and chemical methods ineffective for remediation. Therefore, eco-friendly, long-term, and stable in-situ ecological restoration technologies are urgently needed.

[0003] The submerged plant *Vallisneria natans* is widely distributed and can achieve long-term purification of water bodies by absorbing and accumulating arsenic. It also possesses the function of creating "underwater forests" and improving the aquatic ecological environment, making it an ideal material for in-situ remediation of water bodies with low concentrations of arsenic pollution. However, the remediation of *Vallisneria natans* alone has significant limitations: in low-concentration arsenic environments, *Vallisneria natans* lacks sufficient driving force for arsenic accumulation, resulting in low remediation efficiency; under long-term arsenic exposure, the antioxidant system of *Vallisneria natans* is continuously damaged, making it unable to maintain a long-term stable remediation capacity; and the limited arsenic binding sites on the surface of *Vallisneria natans* rhizomes result in insufficient ability to fix arsenic in the rhizosphere.

[0004] Existing research largely focuses on green algae-assisted phytoremediation, with very little study on *Nyctaginus spp.*, a diatom. *Nyctaginus spp.*, a typical benthic diatom, possesses a siliceous cell wall, strong environmental adaptability, and the ability to stably attach to plant surfaces, making it highly compatible with the habitat of *Vallisneria natans*. However, current techniques have not revealed the specific and stable enhancement mechanism of *Nyctaginus spp.* on arsenic accumulation in *Vallisneria natans*, nor have they developed an engineerable *Nyctaginus spp.*-*Vallisneria natans* symbiotic in-situ remediation system, thus failing to address the core need for long-term stable remediation of water bodies contaminated with low concentrations of arsenic.

[0005] In existing technologies, CN116282568B discloses the purification of polluted water bodies by combining Vallisneria natans with an adsorption layer and photosynthetic bacteria; CN108383243B discloses the enrichment of heavy metals in water bodies by combining Vallisneria natans with Ceratophyllum demersum and Myriophyllum spicatum; CN108101328B discloses the selection of corresponding submerged plants for sediment remediation based on the type of heavy metal. However, none of the above existing technologies disclose a specific symbiotic combination of Vallisneria natans and Vallisneria natans, do not reveal the mechanism by which siliceous cell attachment enhances the stabilization of rhizosphere arsenic, and cannot solve the technical problems of insufficient enrichment driving force of Vallisneria natans under low concentrations of arsenic and unstable long-term in-situ ecological restoration effects, making it difficult to meet the long-term in-situ ecological restoration needs of sensitive water bodies such as drinking water sources.

[0006] Therefore, developing an in-situ remediation method for arsenic pollution based on the symbiotic system of *Rhizophora stylosa* and *Vallisneria natans* has significant practical value. Summary of the Invention

[0007] To address the problems of poor root and rhizome accumulation stability, large long-term fluctuations in effectiveness, and weak adaptability to natural environments in existing methods of remediating arsenic-contaminated waters using only *Vallisneria natans*, this invention provides an in-situ arsenic remediation method based on a symbiotic system of *Nyctaginus spp.* and *Vallisneria natans*. This invention selects *Nyctaginus spp.*, a diatom, utilizing its siliceous cell walls to stably attach to the roots and rhizomes of *Vallisneria natans*, forming a highly compatible symbiotic system. Through the amplification of interfacial binding sites, activation of rhizosphere arsenic speciation, and enhanced stress resistance, the accumulation of arsenic in *Vallisneria natans* is significantly improved, ultimately achieving in-situ ecological remediation of water bodies with low arsenic concentrations.

[0008] Firstly, a method for in-situ remediation of arsenic contamination is provided, including: S1: Plant Vallisneria natans in the restored water system; S2: Inoculate the expanded cultured *Rhizoctonia solani* into the remediation water system to construct a *Rhizoctonia solani*-*Vallisneria natans* symbiotic system; S3: Continuous repair, supplementing with inoculation of *Rhizophora stylosa* and harvesting senescent plants to complete the repair.

[0009] In some embodiments, in S1, Vallisneria natans with a plant height of 25-35cm is selected, and after 15 days of hydroponic adaptation in Hogrange nutrient solution, it is transplanted into the restoration water system for planting.

[0010] In some embodiments, after planting Vallisneria natans in S1, modified Hogland nutrient solution is added for cultivation.

[0011] In some embodiments, the hydroponic culture is adapted to a culture temperature of 28~33 ℃, a pH stable at 6.1~6.5, a daily full-spectrum light duration of ≥8 h, and regular aeration.

[0012] In some embodiments, in S1, Vallisneria natans is planted at a density of 30 to 60 plants per cubic meter of the remediation system, preferably 30, 40, 50, or 60 plants.

[0013] In some embodiments, in S2, *Rhizophora glutinosa* is inoculated at 2-8% (v / v) of the total volume of the remediation system, preferably 2%, 3%, 4%, 5%, 6%, 7%, or 8%.

[0014] In some embodiments, the *Nyctaginus* species were propagated using CSI medium at 23±0.5 ℃, light intensity of 3960 lux, and a light-dark cycle of 12 h:12 h until the logarithmic growth phase was reached. 680 =1.00±0.05.

[0015] In some embodiments, the CSI medium contains Ca(NO3)2·4H2O, KNO3, MgSO4·7H2O, β-glycerophosphate pentahydrate (β-Na2·glycerophosphate·5H2O), and vitamin B. 12 (Vitamin B) 12 Biotin, thiamine HCl, PIV, HEPES, Na2SiO3·9H2O and soil extract; PIV contains Na2EDTA, MnCl2·4H2O, ZnCl2·7H2O, Na2MoO4·2H2O, FeCl3·6H2O and CoCl2·6H2O; The soil extract preparation process is as follows: unfertilized garden soil is dissolved in distilled water, heated, allowed to stand and cool, and then precipitated and filtered. The supernatant is collected; the filtrate is sterilized by high temperature and high pressure to obtain the extract.

[0016] In some embodiments, the CSI medium contains: Ca(NO3)2·4H2O 1 mL / L; KNO3 1 mL / L; MgSO4·7H2O 1 mL / L; β-glycerophosphate disodium pentahydrate 1 mL / L; and vitamin B... 12 The dosages are as follows: 0.1 μg / L for biotin, 10 μg / L for thiamine hydrochloride, 6 mL / L for PIV, 0.5 g / L for HEPES, 0.1 g / L for Na2SiO3·9H2O, 30 mL / L for soil extract, and pH adjusted to 7.0. In PIV, the concentrations were: Na₂EDTA 0.75 g / L dH₂O; MnCl₂·4H₂O 0.041 g / L dH₂O; ZnCl₂·7H₂O 0.005 g / L dH₂O; Na₂MoO₄·2H₂O 0.004 g / L dH₂O; FeCl₃·6H₂O 0.097 g / L dH₂O; and CoCl₂·6H₂O 0.002 g / L dH₂O. The soil extract preparation process is as follows: 200 g of unfertilized garden soil is transferred to a suitable container, 1000 mL of distilled water is added, and the container opening is sealed with a breathable material; the container is placed in a boiling water bath and heated continuously for 3 hours, then allowed to stand, cool, and settle for 24 hours; the above heating-precipitation operation is repeated three times, the mixture is filtered, and the supernatant is collected; the filtrate is sterilized by high temperature and high pressure to obtain the extract.

[0017] In some embodiments, the arsenic concentration in the remediation water system is 10~100 μg / L, preferably 10 μg / L, 20 μg / L, 30 μg / L, 40 μg / L, 50 μg / L, 60 μg / L, 70 μg / L, 80 μg / L, 90 μg / L, or 100 μg / L.

[0018] In some embodiments, in S3, the basic repair cycle for continuous repair is 21 days.

[0019] In some embodiments, in S3, continuous repair culture is carried out at a temperature of 28~33 ℃, with a light exposure of ≥8 h per day, aeration 3 times a day for 1 h each time, and pH stable at 6.1~6.5 for 21 consecutive days. The modified Hogland nutrient solution is replaced every 7 days to maintain the nutritional stability of the system.

[0020] In some embodiments, in S3, *Nyctaginus spp.* solution is replenished every 3 months, and senescent *Vallisneria natans* plants are harvested and new plants are added every 6 months. *Nyctaginus spp.* forms a stable attachment interface on the surface of *Vallisneria natans* rhizomes through its siliceous cell wall, increasing arsenic binding sites in the rhizomes. This symbiotic system can alleviate oxidative damage to *Vallisneria natans* caused by arsenic, maintain chlorophyll content and SOD activity, reduce MDA content, and enhance the environmental adaptability and long-term in-situ ecological restoration stability of *Vallisneria natans*.

[0021] In some embodiments, the modified Hogland nutrient solution has a nitrogen-to-phosphorus molar ratio of 32:1, a nitrate-to-nitrogen ratio of 9:1, and a significant reduction in phosphorus sources. The amount of macroelements such as calcium, magnesium, and potassium is reduced by 55% to lower the total ionic strength, weaken the interference of high salt ions on arsenic detection, and enhance pH buffering capacity to adapt to the characteristics of natural freshwater environments.

[0022] The mechanism by which the *Rhizophora scoparia*-*Vallisneria natans* symbiotic system enhances arsenic accumulation: 1. Amplification of interface sites, increasing arsenic binding sites: Rhizophora glutinosa attaches stably to the surface of the rhizomes and leaves of Vallisneria natans in the form of dispersed siliceous cells, significantly increasing the abundance of polar functional groups such as hydroxyl, amino, carboxyl, and polysaccharide CO on the surface of Vallisneria natans, especially strengthening the arsenic binding sites on the surface of the rhizomes, achieving stable fixation of arsenic in the rhizosphere, and meeting the needs of long-term in-situ remediation.

[0023] 2. Activation of arsenic forms to improve arsenic absorbability: Through metabolic activities, *Nyctaginus glutinosa* regulates rhizosphere pH and secretes extracellular polymers to convert poorly absorbed fixed arsenic into readily absorbable arsenic, thereby reducing the rhizosphere blocking effect of arsenic, improving the bioavailability of arsenic, and making it easier for arsenic to be accumulated by *Nyctaginus glutinosa*.

[0024] 3. Alleviating arsenic stress damage: Rhizoma Nitrariae can stably alleviate the growth inhibition of Vallisneria natans by arsenic, promote root development, delay chlorophyll degradation, maintain the stability of the photosynthetic system, reduce MDA content, maintain SOD activity, reduce oxidative damage under long-term arsenic exposure, and enhance the environmental stress resistance of Vallisneria natans.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention uses *Nyctaginella tamariscina* as the functional algae species to construct a stable attachment symbiotic system with *Vallisneria natans*. *Nyctaginella tamariscina*, with its siliceous cell wall, specifically attaches to the surface of *Vallisneria natans* rhizomes, significantly enhancing the absorption and accumulation of arsenic by *Vallisneria natans* through interfacial binding site amplification, rhizosphere arsenic speciation activation, and physiological stress protection. Under 100 μg / L arsenic stress, the arsenic accumulation stability of *Vallisneria natans* is 15.29% higher than that of *Vallisneria natans* alone, effectively reducing the arsenic concentration in water. This invention solves the problems of low remediation efficiency and susceptibility to arsenic stress damage in low-concentration arsenic water bodies when *Vallisneria natans* is used alone. It is suitable for in-situ ecological remediation of low-concentration arsenic-polluted water bodies such as lakes, reservoirs, and drinking water sources, and has advantages such as stable effects, ecological safety, simple operation and maintenance, and long-lasting effects. Specifically: 1. Stable and long-lasting remediation effect: It has a stable removal effect on water bodies contaminated with arsenic ranging from 10 to 100 μg / L. The arsenic accumulation capacity of Vallisneria natans rhizomes continues to improve without significant fluctuations in effect, making it suitable for long-term in-situ remediation scenarios.

[0026] 2. Strong environmental adaptability: As a benthic diatom, *Rhizophora glutinis* is highly adaptable to natural environmental conditions such as low temperature, low light, and nutrient fluctuations. It can form a stable symbiotic system with *Vallisneria natans* in complex water bodies in the field, solving the problem of poor field application effect of laboratory systems.

[0027] 3. Excellent ecological compatibility: It is a pure biological symbiotic system that does not damage the original ecological structure of the water body. At the same time, it can build underwater vegetation and optimize benthic habitats. It has the dual functions of arsenic pollution remediation and aquatic ecosystem restoration, and is suitable for ecologically sensitive water bodies such as drinking water sources and lakes.

[0028] 4. Extremely low operation and maintenance costs: Once the system is built, it can operate stably for a long time. Only aging plants need to be harvested periodically and a small amount of algae seeds need to be added. No complicated equipment or continuous chemical addition is required, making it suitable for large-scale restoration of large water areas.

[0029] 5. No risk of secondary pollution: Arsenic is mainly fixed in the rhizomes and algal cells of Vallisneria natans. Arsenic can be safely removed through plant harvesting, leaving no chemical residues and eliminating the risk of secondary pollution.

[0030] Terminology Explanation Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0031] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0032] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0033] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0034] In the following content, all figures disclosed herein, whether or not the words "approximately" or "about" are used, are approximate values. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%, etc. Whenever a figure with a value of N is disclosed, any figure with a value of N±1%, N±2%, N±3%, N±5%, N±7%, N±8%, N±10%, N±15%, or N±20% will be explicitly disclosed, where "±" indicates addition or subtraction. Attached Figure Description

[0035] Figure 1 This is a process flow diagram of the symbiotic system in Example 1.

[0036] Figure 2 This is a SEM image of *Rhizoctonia solani* attached to the surface of the rhizome of *Vallisneria natans*.

[0037] Figure 3 The graphs show the long-term changes in arsenic concentration in water bodies for different treatment groups. (a): Arsenic concentration 10 μg / L; (b): Arsenic concentration 50 μg / L; (c): Arsenic concentration 100 μg / L.

[0038] Figure 4 The infrared spectra of rhizomes of *Vallisneria natans* cultured for 14 days under 100 μg / L arsenic stress are shown. The blue curve represents the *Vallisneria natans* cultured alone, and the red curve represents the *Vallisneria natans* co-cultured with *Rhizophora stylosa*.

[0039] Figure 5 Infrared spectra of leaves of *Vallisneria natans* cultured for 14 days under 100 μg / L arsenic stress. The blue curve represents the *Vallisneria natans* cultured alone, and the red curve represents the *Vallisneria natans* co-cultured with *Nyctaginus spp.* Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0041] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0042] In this invention, room temperature / normal temperature refers to 20~30℃, preferably 25~30℃.

[0043] Example 1: In-situ ecological remediation of 100 μg / L arsenic-contaminated water 1. Pretreatment of Vallisneria natans: Select Vallisneria natans with a plant height of 30 cm and well-developed root system. After washing with deionized water, transfer them to a culture device containing 1 / 2 Hoagland nutrient solution and allow them to adapt to hydroponics for 15 days before use. Culture environment conditions: temperature 28~33℃, daily full-spectrum light duration ≥8 h, aeration 3 times a day for 1 h each time, and nutrient solution pH stable at 6.1~6.5.

[0044] 2. Rhomboid algae ( Nitzschia palea Expansion culture: Using CSI medium, the algae were expanded in an artificial climate incubator at a temperature of 23±0.5 ℃, a light intensity of 3960 lux, and a light-dark cycle of 12 h:12 h until the algal solution reached its OD value. 680=1.00±0.05 logarithmic growth period, for future use.

[0045] Table 1. Formulation of CSI medium

[0046] Adjust the pH of the CSI medium to 7.0.

[0047] The soil extract was prepared as follows: 200 g of unfertilized garden soil was weighed and transferred to a suitable container. 1000 mL of distilled water was added, and the container was sealed with a breathable material. The container was placed in a boiling water bath and heated continuously for 3 hours, followed by standing to cool and settle for 24 hours. This heating-precipitation process was repeated three times. The mixture was then filtered, and the supernatant was collected. The filtrate was sterilized by high-temperature and high-pressure sterilization and then stored at 4°C for later use.

[0048] Table 2 PIV Components Table

[0049] 3. System Construction: (1) Symbiotic system group: 5 cm glass beads were laid in a glass tank culture device (67 cm × 45 cm × 35.5 cm), 5 pretreated Vallisneria natans were planted, and modified Hogland nutrient solution was added (the nitrogen-phosphorus molar ratio was adjusted to 32:1, the nitrate-ammonium nitrogen ratio was set to 9:1 and the phosphorus source was significantly reduced, and the amount of macroelements such as calcium, magnesium and potassium was reduced by 55% to reduce the total ionic strength, weaken the interference of high salt ions on arsenic detection, and at the same time strengthen the pH buffering capacity to adapt to the characteristics of natural freshwater environment); 5% (v / v) of the total volume of the remediation system was inoculated with logarithmic growth phase of Rhizoctonia solani solution to construct Rhizoctonia solani-Vallisneria natans symbiotic remediation system, and the arsenic concentration in the water to be remediated was 100 μg / L.

[0050] (2) Single Vallisneria group: without inoculation with Rhizophora branii. The rest are the same as (1).

[0051] (3) Algae-free control group: No Rhomboidia glutinosa was inoculated, and no Vallisneria natans was planted. The rest was the same as (1).

[0052] (4) Single Rhizophora glutinosa group: Only Rhizophora glutinosa was inoculated in the water. The rest were the same as (1).

[0053] (5) Common green algae-Vallisneria group: Common green algae were inoculated to replace the inoculated Rhizophora scoparia in the water. The rest is the same as (1).

[0054] 4. Repair culture: Temperature 28~33 ℃, light ≥8 h per day, aeration 3 times a day for 1 h each time, pH stable at 6.1~6.5, culture continuously for 21 days, change the modified Hogland nutrient solution every 7 days to maintain the nutritional stability of the system.

[0055] 5. Repair effect: such as Figure 3 As shown in (c), after 21 days of cultivation, the residual arsenic concentration in the water of the symbiotic system group was 29.96% lower than that of the single Vallisneria natans group; the overall arsenic content of Vallisneria natans in the symbiotic system group was 15.29% higher than that of the single Vallisneria natans group, and the arsenic content of Vallisneria natans in the symbiotic system group increased steadily; the arsenic accumulation in the Vallisneria natans rhizomes of the single Vallisneria natans group was low, and the long-term operation showed obvious fluctuations, and the in-situ ecological restoration efficiency under low arsenic concentration continued to decline. After 21 days of cultivation, the residual arsenic concentration in the water of the symbiotic system group was 20.54% lower than that of the single *Nyctaginea* group; the overall arsenic content of *Vallisneria natans* in the symbiotic system group was 16.37% higher than that of the single *Nyctaginea* group. like Figure 2 As shown, the common green algae-Vallisneria natans combination system cannot form the silica cell attachment interface unique to Rhizophora tataricus, the arsenic enrichment stability is improved by less than 5%, and there is no significant arsenic remediation synergy between algae and grass. In the symbiotic system, *Rhizophora stearensis* specifically adhered to the surface of *Vallisneria natans* rhizomes using its siliceous cell walls, resulting in a 15.29% increase in arsenic accumulation stability, a 20% increase in *Vallisneria natans* plant height, a significant increase in biomass, a maximum increase of 9.57% in leaf SPAD value, an 8.44% decrease in MDA content, and a 7.68% increase in SOD activity, achieving stable and coordinated repair.

[0056] like Figure 4 and Figure 5 The images shown are Fourier transform infrared spectra of *Vallisneria natans* leaves and rhizomes in a single *Vallisneria natans* group and a *Vallisneria natans*-*Rhizophora spp.* symbiotic system. Compared to the single *Vallisneria natans* group, the *Vallisneria natans* leaves and rhizomes in the symbiotic system have significantly different wavelengths (3418–3422 cm⁻¹). - ¹Near hydroxyl characteristic peak, 1627~1642 cm⁻¹ - ¹Near the characteristic peak of amide C=O, 1034–1321 cm⁻¹ - ¹The absorption intensity of the CO characteristic peak of polysaccharides in the interval was significantly enhanced, indicating that after the colonization of *Rhizophora brasiliensis*, it can induce the generation of a large number of active functional groups of hydroxyl, amino and polysaccharide on the surface of *Vallisneria natans* rhizomes and leaves. The abundant active functional groups can provide more complexation and adsorption sites for arsenic ions, revealing the intrinsic mechanism of synergistic enrichment and fixation of arsenic in the algae-grass symbiotic system at the micro-interface level.

[0057] Example 2: In-situ ecological restoration of water bodies contaminated with arsenic at a concentration of 50 μg / L 1. The pretreatment of Vallisneria natans is the same as in Example 1.

[0058] 2. The propagation of *Rhizophora stylosa* was carried out in the same manner as in Example 1, and cultured until OD... 680 =0.99, reserved.

[0059] 3. System construction: (1) Symbiotic system group, (2) Single Vallisneria group, (3) Algae-free control group, and (4) Single Rhizophora scoparia group are all the same as in Example 1, except that the arsenic concentration in the water is 50 μg / L.

[0060] 4. The culture conditions for repair are the same as in Example 1.

[0061] 5. Repair effect: such as Figure 3 As shown in (b), after 21 days of cultivation, the residual arsenic concentration in the water of the symbiotic system group was 32.86% lower than that of the single Vallisneria natans group; the residual arsenic concentration in the water of the symbiotic system group was 35.79% lower than that of the single Rhizophora scoparia group; the overall arsenic content of Vallisneria natans in the symbiotic system group was 14.81% higher than that of the control group without algae, and Vallisneria natans showed excellent growth and no signs of arsenic stress damage.

[0062] The results show that the present invention still possesses stable in-situ ecological restoration capabilities that are superior to existing technologies in water bodies contaminated with low to medium concentrations of arsenic.

[0063] Example 3: Long-term in-situ ecological restoration of water bodies contaminated with low concentrations of 10 μg / L arsenic 1. The pretreatment of Vallisneria natans is the same as in Example 1.

[0064] 2. The propagation of *Rhizoctonia solani* was carried out in the same manner as in Example 1.

[0065] 3. System construction: (1) Symbiotic system group: Simulate the in-situ remediation scenario of a lake, plant Vallisneria natans in a 100 L simulated water device, and inoculate with Rhizoctonia solani at a 5% inoculation rate. The arsenic concentration in the water is 10 μg / L. (3) The algae-free control group was constructed under the same conditions as (1) symbiotic system group, except that no plants were inoculated.

[0066] 4. Long-term cultivation: Cultivate continuously for 6 months under natural light and room temperature conditions, supplement with Rhizophora glutinosa every 3 months, and harvest aged Eriocaulon buergerianum every 6 months.

[0067] 5. Repair effect: such as Figure 3 As shown in (a), compared with the algae-free control group, the arsenic concentration in the water of the symbiotic system group remained consistently below 2 μg / L within 6 months of cultivation; Vallisneria natans continued to grow and formed a stable community, with the arsenic accumulation in the rhizomes remaining consistently above 1.0 mg / kg; ecological indicators such as water transparency and dissolved oxygen were significantly improved, and there were no ecological risks such as algal blooms.

[0068] The results of six months of long-term operation have proven that this invention can achieve long-term in-situ ecological restoration of water bodies with low concentrations of arsenic, fully meeting the restoration needs of ecologically sensitive water bodies such as water sources.

[0069] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A method for in-situ remediation of arsenic contamination, characterized in that, Constructing a symbiotic system of *Nyctaginosa* and *Vallisneria natans* to achieve in-situ water body remediation includes: S1: Plant Vallisneria natans in the restored water system; S2: Inoculate the expanded cultured *Rhizoctonia solani* into the remediation water system to construct a *Rhizoctonia solani*-*Vallisneria natans* symbiotic system; S3: Continuous repair, supplementing with inoculation of *Rhizophora stylosa* and harvesting senescent plants to complete the repair.

2. The repair method according to claim 1, characterized in that, In S1, select Vallisneria natans with a plant height of 25-35cm, acclimate them to hydroponics in Hogland nutrient solution for 15 days, and then transplant them into the restoration water system for planting. Alternatively, after planting Vallisneria natans in S1, add a modified Hogland nutrient solution for cultivation.

3. The repair method according to claim 2, characterized in that, The hydroponic culture is adapted to a temperature of 28~33 ℃, a pH of 6.1~6.5, a daily full-spectrum light duration of ≥8 h, and regular aeration; or, in the modified Hogland nutrient solution, the nitrogen-phosphorus molar ratio is adjusted to 32:1 and the ammonium nitrate-nitrogen ratio is set to 9:

1.

4. The repair method according to claim 1, characterized in that, In S1, Vallisneria natans is planted at a density of 30-60 plants per cubic meter of the remediation system. Alternatively, in S2, inoculate *Rhizophora glutinosa* at 2-8% (v / v) of the total volume of the remediation system.

5. The repair method according to claim 1, characterized in that, The *Rhizoctonia solani* was propagated using CSI medium at 23±0.5 ℃, light intensity of 3960 lux, and a light-dark cycle of 12 h:12 h until the logarithmic growth phase. OD... 680 =1.00±0.

05.

6. The repair method according to claim 5, characterized in that, In the CSI medium, the concentrations of Ca(NO3)2·4H2O, KNO3, MgSO4·7H2O, and β-glycerophosphate disodium pentahydrate were 1 mL / L; vitamin B1 was also present. 12 The dosages are as follows: 0.1 μg / L for biotin, 10 μg / L for thiamine hydrochloride, 6 mL / L for PIV, 0.5 g / L for HEPES, 0.1 g / L for Na2SiO3·9H2O, 30 mL / L for soil extract, and pH adjusted to 7.

0. In PIV, the concentrations of Na₂EDTA, MnCl₂·4H₂O, ZnCl₂·7H₂O, Na₂MoO₄·2H₂O, FeCl₃·6H₂O, and CoCl₂·6H₂O were 0.005 g / L dH₂O, 0.004 g / L dH₂O, 0.097 g / L dH₂O, and 0.002 g / L dH₂O. The soil extract preparation process is as follows: 200 g of unfertilized garden soil is transferred to a suitable container, 1000 mL of distilled water is added, and the container opening is sealed with a breathable material; the container is placed in a boiling water bath and heated continuously for 3 hours, then allowed to stand, cool, and settle for 24 hours; the above heating-precipitation operation is repeated three times, the mixture is filtered, and the supernatant is collected; the filtrate is sterilized by high temperature and high pressure to obtain the extract.

7. The repair method according to claim 1, characterized in that, The arsenic concentration in the remediation water system is 10~100 μg / L.

8. The repair method according to claim 1, characterized in that, In S3, the basic repair cycle for continuous repair is 21 days.

9. The repair method according to claim 1, characterized in that, In S3, continuous repair culture was carried out at a temperature of 28~33 ℃, with a light exposure of ≥8 h per day, aeration 3 times a day for 1 h each time, and pH stable at 6.1~6.5 for 21 consecutive days. The modified Hogland nutrient solution was replaced every 7 days to maintain the nutritional stability of the system.

10. The repair method according to claim 1, characterized in that, In S3, the plant is inoculated with Rhizophora glutinosa solution every 3 months, and old plants are harvested and new plants are added every 6 months.

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