Submerged plant-ecological interface system for in-situ remediation of water bodies and preparation method and use thereof

Abstract

The invention provides a submerged plant-ecological interface system for in-situ treatment of water bodies, which provides a functional microbial community and fertilizer for the growth of submerged plants, so that the submerged plants are rooted in the ecological interface to form a plant rhizosphere microbial environment with the ecological interface, and a submerged plant-ecological interface system is constructed, which can effectively improve the problems of high planting environment requirement, large construction difficulty, low planting survival rate, slow water purification effect, high maintenance cost and the like faced by the existing submerged plant purification water body; the plants and microorganisms are organically combined together to form an efficient biological repair composite, which has a significant pollution removal, water purification and water body ecological restoration effect, and has a good application prospect in the field of water pollution treatment.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection technology and relates to a submerged plant-ecological interface system for in-situ treatment of polluted rivers, lakes, ponds and other water bodies, and its preparation method. Background Technology

[0002] Nitrogen and phosphorus pollution in water bodies, along with river siltation, have caused severe degradation of ecosystems. The application of plants and microorganisms for bio-enhanced water pollution control and ecological restoration is a commonly used technique. Among these, using submerged plants to construct underwater forests for in-situ remediation not only purifies water bodies and improves bottom sediment but also facilitates ecological restoration and landscape creation, thus having a wide range of applications.

[0003] The survival rate of submerged plants is a significant challenge, as they have high requirements for the aquatic environment. Current methods for planting submerged plants without water require draining the water body, which is time-consuming and labor-intensive, and it's difficult to remedy the situation if the plants fail. Planting in water presents numerous difficulties. Submerged plants require a relatively stable aquatic substrate, primarily composed of mud and sand. Hard, heavily polluted substrates are unsuitable for their survival. A common method is to dredge the substrate before planting, but this is costly, and residual pollutants after dredging can also negatively impact the planting and growth of submerged plants.

[0004] A stable substrate environment is an important condition for the cultivation of submerged plants. Therefore, constructing submerged plants planted at the ecological interface is easy to plant in situ and has a high survival rate. The resulting mixture of substrate and plants has a large number of microorganisms growing. Organically combining plants and microorganisms to form a highly efficient bioremediation complex and a biological treatment system will have significant effects on pollution removal, water purification and aquatic ecological restoration, and can effectively solve existing problems. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a submerged plant-ecological interface system for in-situ water remediation. By providing submerged plants with functional microbial communities and fertilizers to regulate their growth, the submerged plants take root at the ecological interface, forming a rhizosphere microbial environment with the ecological interface, creating a stable complex, and constructing an ecological interface system. This system enables the rapid planting of submerged plants in the water body to be remediated, the sealing of bottom sediment, and the in-situ remediation of water quality and ecology.

[0006] The submerged plant-ecological interface system of this invention consists of a carpet-like material, namely an ecological interface, made of submerged plants and natural woody fibrous materials. The submerged plants take root and grow on the ecological interface, and the root system and the ecological interface are integrated. During the preparation process, functional microorganisms, especially immobilized functional microorganisms, are used to regulate the microbial community attached after the submerged plant roots and the ecological interface are integrated, as well as the microbial community of the biofilm formed, so that it has specific ecological functions.

[0007] On one hand, the present invention provides a submerged plant-ecological interface system, comprising submerged plants, an ecological interface, and a mud mixture; the ecological interface is a blanket-like material processed from woody filaments; the mud mixture contains immobilized microorganisms, the immobilized microorganisms comprising microorganisms and carriers encapsulating the microorganisms.

[0008] Furthermore, the mud mixture also contains magnesium ammonium phosphate.

[0009] Further, the mud mixture comprises 4.0-5.0 parts water, 4.0-5.0 parts bottom mud, 0.5-1.0 parts magnesium ammonium phosphate, and 0.5-1.0 parts immobilized microorganisms.

[0010] Furthermore, when used in nitrogen-polluted water bodies, the microorganisms include nitrifying microorganisms and / or denitrifying microorganisms; when used in polluted water bodies with excessive COD, the microorganisms include Bacillus pumilus and / or Enterobacteriaceae.

[0011] Furthermore, when used in nitrogen-polluted water bodies, the microorganisms include 2.5 parts nitrifying microorganisms and 7.5 parts denitrifying microorganisms, with a total viable count ≥ 10 billion CFU / g; when used in water bodies with excessive COD, the microorganisms include 5.5 parts Bacillus pumilus and 4.5 parts Enterobacteriaceae, with a total viable count ≥ 10 billion CFU / g.

[0012] Furthermore, the carrier for loading microorganisms includes sodium alginate, agar, diatomaceous earth, and CaCl2.

[0013] Furthermore, the submerged plant is Vallisneria seeds; the ecological interface is a coconut fiber mat with a thickness of 1.5–3 cm; the Vallisneria seeds are spread on the surface of the coconut fiber mat; the mud mixture covers the coconut fiber mat and the Vallisneria seeds with a thickness of 1–2 mm.

[0014] As a preferred option, in the preparation of the submerged plant-ecological interface system, the submerged plant is commercially available Vallisneria seeds; the ecological interface is a commercially available coconut fiber mat with a thickness of 1.5-3 cm, formed by hot pressing natural coconut fiber with added cross-linking agent and hydrophilic additives, which can sink to the bottom of the water after complete immersion; the preparation method of the immobilized microbial particles is as follows: the coating system contains 1 part of 4% sodium alginate water solution, 1 part of 2.5%-3.2% agar aqueous solution, mixed and heated in a water bath at 60℃, 0.6 parts of diatomaceous earth are added and stirred evenly, and then cooled. Add 0.2-0.4 parts of the compound bacterial agent to a temperature below 50℃, mix further, pour into a mold, allow to cool naturally, and then immerse in a 4% CaCl2 aqueous solution for 4 hours to solidify. Finally, wash with deionized water, drain, and cut into 5*5*5mm immobilized functional microbial particles for later use. The composition of the compound bacterial agent is determined according to the water pollution status of the submerged plant-ecological interface system to be applied. For common nitrogen-polluted water bodies, the preferred composition of the compound bacterial agent is Nitrosomonas NE-1. The bacterial powder prepared from the combination of *Europaea NE-1* (China Center for Type Culture Collection No.: CCTCC NO: M2019754) and *Pseudomonas stutzeri* ZH-14 (China Center for Type Culture Collection No.: CCTCC NO: M2018730) with aerobic denitrification function has a total viable count of ≥10 billion CFU / g. The compound bacteria contain 2.5 parts of *Europaea NE-1* and 7.5 parts of *Pseudomonas stutzeri* ZH-14. The microorganisms in this compound bacteria have nitrification and aerobic denitrification functions. They can also be replaced by other commercially available qualified products with nitrification and aerobic denitrification functions and a total viable count of ≥10 billion CFU / g. The compound ratio is also 1:3. For polluted water bodies with common COD exceeding the standard, it is composed of Bacillus pumilus BSK-9 (China Center for Type Culture Collection No.: CCTCC NO: M2017221) and Enterobacter sp. AOZ-1 (China Center for Type Culture Collection No.: CCTCC NO: M2017219). The total viable count of the prepared bacterial powder is ≥50 billion CFU / g. The compound bacteria contains 5.5 parts of BSK-9 and 4.5 parts of AOZ-1.

[0015] On the other hand, the present invention provides a method for preparing the submerged plant-ecological interface system as described above, the method comprising the following steps:

[0016] 1) Soak the seeds of the bitter grass in clean water and cultivate them until they sprout white buds;

[0017] 2) Soak the ecological interface with clean water, then place the ecological interface in a container to keep it moist.

[0018] 3) Spread the germinated white shoots of Vallisneria seeds on the surface of the ecological interface, cover with mud mixture, and add water to be restored or clean water to the thickness of the ecological interface.

[0019] 4) Once the seedlings have emerged, increase the water level to submerge them until the stems and leaves of the Vallisneria natans grow to 7-9 cm and the roots penetrate the ecological interface. The seedlings are then ready for use.

[0020] In some methods, the preparation steps of the submerged plant-ecological interface system are as follows:

[0021] 1) Soak the seeds of Vallisneria natans in clean water and cultivate them at 20℃ until they germinate into white buds;

[0022] 2) Soak the ecological interface with clean water, and keep about 1cm of water in the container holding the ecological interface to keep the ecological interface moist.

[0023] 3) Spread the germinated white-budded seeds of Vallisneria natans evenly on the surface of the ecological interface, and cover them with a 1-2 mm thick mud mixture, the composition of which is: 4.0-5.0 parts water, 4.0-5.0 parts bottom mud to be used for water body remediation, 0.5-1.0 parts magnesium ammonium phosphate, and 0.5-1.0 parts immobilized microorganisms. After placing it at 20-25℃ for 24 hours, add the water to be used for water body remediation or clean water to the thickness of the ecological interface.

[0024] 4) After 3-5 days, once the seedlings have emerged, increase the water level to submerge them until the stems and leaves of the Vallisneria natans reach 5cm in length and the roots penetrate the ecological interface. The seedlings are then ready for use.

[0025] The prepared submerged plant-ecological interface can be used for in-situ remediation of polluted rivers, lakes, ponds and other water bodies. It can be directly removed from the cultivation device and laid on the water body to be remediated.

[0026] On another aspect, the present invention provides a method for in-situ remediation of water bodies, which mainly involves removing the submerged plant-ecological interface system prepared by the method described above from the cultivation device and laying it on the bottom of the water body to be remediated.

[0027] In another aspect, the present invention provides the use of a mud mixture for preparing a submerged plant-ecological interface system, wherein the mud mixture contains immobilized microorganisms and magnesium ammonium phosphate.

[0028] The beneficial effects of this invention are as follows: 1) The prepared submerged plant-ecological interface becomes a submerged plant-microorganism complex that can be easily moved and used, forming a unique biochemical system. Through plant growth and nutrient absorption, it improves water quality and increases dissolved oxygen in the water. The plant rhizosphere microbial environment formed by the roots in the ecological interface and the microbial community attached to the substrate form a biofilm with efficient pollutant degradation and transformation capabilities. After covering the bottom sediment, it forms a biochemical isolation layer at the sediment-water interface with efficient material transformation and energy flow regulation capabilities, effectively reducing the release of endogenous substances in the water, thereby achieving the beneficial effects of water pollution remediation and ecological restoration; 2) The prepared submerged plant-ecological interface naturally forms a stable complex. It can be transplanted to various natural water bodies for in-situ remediation with low requirements for the bottom sediment environment. Most water bodies do not require dredging. It is easy to construct, has a high survival rate, works quickly, has a long-lasting effect, is easy to maintain, and has low cost. 3) After the application of this submerged plant-ecological interface in the water body, a stable environment with plants and microorganisms as the main body is formed at the bottom, which is conducive to the growth of protozoa and metazoa, thereby promoting the formation of the aquatic food chain and the restoration of the aquatic ecology.

[0029] Therefore, the submerged plant-ecological interface prepared by this invention can effectively improve the existing problems of high planting environment requirements, difficult construction, low survival rate, slow water purification effect, and high maintenance cost when using submerged plants to purify water bodies. It organically combines plants and microorganisms to form a highly efficient bioremediation complex, which has significant effects on pollution removal, water purification and water body ecological restoration, and has good application prospects in the field of water pollution control. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the preparation process of the Vallisneria natans-ecological interface system in Example 1;

[0031] Figure 2 This is a scanning electron microscope image of the Vallisneria natans-ecological interface in Example 1;

[0032] Figure 3 This is a schematic diagram showing the treatment effect of medium-concentration ammonia nitrogen wastewater in Experiment Example 1.1.1;

[0033] Figure 4 This is a schematic diagram showing the treatment effect of high-concentration ammonia nitrogen wastewater in Example 1.1.2;

[0034] Figure 5 This is the dilution curve from Example 2.1.1;

[0035] Figure 6 This is a bar chart showing the bacterial community structure and relative abundance (phylum level) of Experiment 2.1.2;

[0036] Figure 7This is a bar chart showing the bacterial community structure composition and relative abundance (genus level) for Experiment 2.1.2;

[0037] Figure 8 The PCoA analysis diagram of the microbial community in Experiment 2.1.3 is shown below;

[0038] Figure 9 This is a dilution curve of the denitrifying bacteria in Example 2.2.1;

[0039] Figure 10 This is a bar chart showing the community structure and relative abundance (phylum level) of denitrifying bacteria in Experiment Example 2.2.2;

[0040] Figure 11 This is a bar chart showing the community structure and relative abundance (genus level) of denitrifying bacteria in Experiment Example 2.2.2;

[0041] Figure 12 The PCoA analysis diagram shows the denitrifying microbial community in Experiment 2.2.3. Detailed Implementation

[0042] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and are not intended to limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0043] Example: Preparation of submerged plant-ecological interface system

[0044] Example 1

[0045] 1.1 Preparation of materials

[0046] The seeds of Vallisneria natans were purchased from Nanjing Tianfu Aquatic Plant Planting Professional Cooperative, and the ecological interface was purchased from Shanghai Muying Ecological Technology Co., Ltd., with a thickness of approximately 2.0 cm. The compound microbial agent was prepared as follows: European Nitrifying Monotrophus NE-1 and Pseudomonas stutzeri ZH-14 bacterial powders processed by Wuhan Kenuo Biotechnology Co., Ltd. were mixed in a 1:3 ratio. The prepared coating system contained 1 part of 4% sodium alginate water solution and 1 part of 3.0% agar water solution. After mixing, the mixture was heated in a water bath at 60°C. 0.6 parts of diatomaceous earth were added and stirred evenly. After cooling to below 50°C, 0.2-0.4 parts of the compound microbial agent were added, further mixed, poured into a mold, and allowed to cool naturally. After solidification, the mixture was immersed in a 4% CaCl2 water solution for 4 hours. Finally, it was washed with deionized water, drained, and cut into 5*5*5mm immobilized functional microbial particles for later use.

[0047] 1.2 Preparation of Vallisneria natans-ecological interface

[0048] 1) Soak the purchased seeds of Vallisneria natans in clean water and cultivate them at 20℃ until they germinate into white buds;

[0049] 2) Place the eco-interface in the tray, moisten it with clean water, and leave about 1cm of water in the tray to keep the eco-interface moist;

[0050] 3) Spread the germinated white-budded seeds of Vallisneria natans evenly on the surface of the ecological interface, and cover them with a 1mm thick mud mixture, which consists of: 4.0 parts water, 5.0 parts bottom mud collected from the Qianjiahe section of the Hangzhou Academy of Agricultural Sciences, 0.5 parts magnesium ammonium phosphate, and 0.5 parts immobilized microorganisms. Mix them evenly before use. After placing them at 20-25℃ for 24 hours, add Qianjiahe River water to a depth of 2.0cm, which is equivalent to the thickness of the ecological interface in the tray.

[0051] 4) After 3-5 days, once the seedlings have emerged, increase the water level to submerge them until the stems and leaves of the Vallisneria natans reach 7-9 cm in length. Check the root system; if it has penetrated the ecological interface, the preparation is complete. A schematic diagram of the preparation process is shown below. Figure 1 As shown.

[0052] 1.3 Scanning electron microscopy observation of the Vallisneria natans-ecological interface

[0053] The prepared Vallisneria natans-ecological interface was observed using scanning electron microscopy on the leaf surface, root surface, and ecological interface surface of Vallisneria natans. Figure 2 The leaves of *Vallisneria natans* are covered with a large number of microorganisms, and biofilms gradually form on the roots, creating a biofilm on the ecological interface. In this embodiment, the roots of submerged plants are closely integrated with the ecological interface (the roots of *Vallisneria natans* grow densely in the ecological interface and are connected as one). The rhizosphere microorganisms and the biofilm formed on the ecological interface together form a microbial system with degradation capabilities.

[0054] Example 2

[0055] The preparation method in this embodiment is the same as that in Example 1, except that the amount of magnesium ammonium phosphate in step 1.2 of this embodiment is 0.8 parts, and the total length of the seedlings at the Vallisneria natans-ecological interface is measured to be 12.5±1.8cm.

[0056] Example 3

[0057] The preparation method in this embodiment is the same as that in Example 1, except that the amount of magnesium ammonium phosphate in step 1.2 of this embodiment is 1 part, and the total length of the seedlings at the Vallisneria natans-ecological interface is measured to be 13.0±1.5cm.

[0058] Example 4

[0059] The preparation method in this embodiment is the same as that in Example 1. The difference is that step 1.2 of this embodiment does not contain magnesium ammonium phosphate, and the total length of the seedlings at the Vallisneria natans-ecological interface is measured to be 9.5±1.2cm.

[0060] Experiment Example 1: The Purification Effect of Submerged Plant-Ecological Interface on Nitrogen-Polluted Water

[0061] 1.1 Effects of submerged plant-ecological interface on nitrogen release and microorganisms in simulated sediment

[0062] Experimental samples: This experiment includes 6 groups, namely blank control group (CK), immobilized microbial group (G), Vallisneria natans group (V), Vallisneria natans + immobilized microbial group (VG), namely the submerged plant-ecological interface system prepared in Example 1, Vallisneria natans + free bacteria group (VF), and free bacteria group (F). Each group has 3 replicates. The ecological interface of each group is the same, and the difference lies in the plants and microorganisms.

[0063] Experimental Method: Polluted river water (Qianjia River, a suburban river in Hangzhou, Zhejiang Province) was collected in a specific container. After measuring the initial water sample parameters, the initial ammonia nitrogen concentration was adjusted with NH4Cl to approximately 2–2.5 mg / L (medium concentration) and 4–5 mg / L (high concentration). The samples were then aliquoted into individual water tanks. Samples were taken daily using 50 mL centrifuge tubes, and the total nitrogen (TN) and NH4+ levels in the water were measured. + -N, NO3 - -N, TOC, and DO were continuously measured for 7-10 days, and the ecological interface area used in the experiment was S = 0.217 m². 2 .

[0064] After the experiment, taking the entire ecological interface—Vallisneria natans—as a whole, the average daily ammonia nitrogen removal per square meter, M, was estimated. AN (mg·m -2 ·d -1 Total nitrogen removal M TN (mg·m -2 ·d -1 ).

[0065] M AN Or M TN =(C0-C S V / ST

[0066] C0 represents the initial concentration of ammonia nitrogen or total nitrogen in the water (mg·L⁻¹). -1 ); C S The concentration of ammonia nitrogen or total nitrogen in the water after the experiment (mg·L) -1 V represents the volume of water used in the experiment (L); S represents the ecological interface area (m²). 2 T represents the processing time (d).

[0067] 1.1.1 Treatment effect of medium-concentration ammonia nitrogen wastewater

[0068] Experimental Procedure: The total volume of water used in each tank for the combined immobilized microorganisms in the Vallisneria natans ecological interface was 35L. River water was collected and regulated with ammonium chloride. After regulation, the ammonia nitrogen was 2.1–2.26 mg / L, nitrate nitrogen was 1.15–1.47 mg / L, total nitrogen was 3.67–3.73 mg / L, and dissolved oxygen was 8.38 mg / L. The experimental temperature was 20℃.

[0069] Results analysis: After 8 days of culture, the results are as follows: Figure 3 As shown, the addition of immobilized microorganisms led to a slight increase in ammonia nitrogen, ultimately resulting in a slight increase in ammonia nitrogen compared to the CK (blank control) group, but the difference between the free group and the CK group was not significant. The ammonia nitrogen removal rate of the *Vallisneria natans* + immobilized microorganism group reached 97.3%. Nitrate nitrogen in all groups initially increased and then decreased, but the blank group and the free bacteria group showed a continuous upward trend. The group with immobilized microorganisms showed a slower increase in nitrate nitrogen and a downward trend, because less ammonia nitrogen was converted, and the bacteria's own utilization or denitrification was stronger. The addition of immobilized microorganisms significantly improved TN removal in the water compared to the CK and free groups. The *Vallisneria natans* + immobilized group (84.2%) also showed stronger TN removal than *Vallisneria natans* (69.6%) and the *Vallisneria natans* + free bacteria group (63.7%), indicating that the addition of immobilized microorganisms enhanced denitrification to some extent. Considering the changes in dissolved oxygen, immobilized microorganisms caused a rapid decrease in dissolved oxygen, thus facilitating denitrification. In summary, under medium concentration conditions, the submerged plant-ecological interface treatment of nitrogen-polluted water can significantly improve the nitrogen removal rate and achieve water purification.

[0070] 1.1.2 Treatment effect of high-concentration ammonia nitrogen wastewater

[0071] Experimental procedure: The water volume in each tank was 35L. River water was collected and adjusted with ammonium chloride. After adjustment, the ammonia nitrogen was 4.18-4.47 mg / L, the nitrate nitrogen was 1.19-1.34 mg / L, the total nitrogen was 5.38-5.64 mg / L, and the dissolved oxygen was 8.65-9.45 mg / L. The experimental temperature was 20℃.

[0072] Results analysis: After 8 days of culture, the results are as follows: Figure 4As shown in the figure, in the high-concentration experiment, the ammonia nitrogen removal rate of the *Vallisneria natans* ecological interface + immobilized microbial community (82.6%) was significantly higher than that of the *Vallisneria natans* ecological interface group (63.4%). The addition of immobilized microorganisms significantly enhanced TN removal in the water compared to the CK (blank control) group and the free microbial group. The *Vallisneria natans* + immobilized microbial community (62.4%) also demonstrated stronger TN removal than *Vallisneria natans* (45.9%) and the *Vallisneria natans* + free microbial community (48.6%), indicating that the addition of immobilized microorganisms enhanced denitrification in the water to some extent. Compared with the results of the medium-concentration experiment, the total nitrogen removal was significantly increased. Overall, under high-concentration conditions, the submerged plant-ecological interface treatment of nitrogen-polluted water still achieves a strong ammonia nitrogen removal effect, unaffected by environmental factors.

[0073] Example 2: High-throughput sequencing analysis of microbial community changes during the treatment of nitrogen-polluted water bodies with different concentrations using a submerged plant-ecological interface system.

[0074] Experimental sample:

[0075] During the experiment of treating water bodies with different concentrations of nitrogen pollution using a submerged plant-ecological interface system, 200 mL water samples were collected every 2 days, filtered through a 0.22 μm filter membrane, and microorganisms were collected for sequencing analysis. The samples from each group are shown in Tables 1 and 2.

[0076] Table 1: Samples from the submerged plant-ecological interface system under medium concentrations of ammonia nitrogen

[0077]

[0078] Table 2: Samples from the submerged plant-ecological interface system under high concentrations of ammonia nitrogen

[0079]

[0080] 2.1 Analysis of bacterial 16S rDNA gene diversity

[0081] 2.1.1 Bacterial Community Alpha Diversity Analysis

[0082] High-throughput sequencing was used to analyze the changes in bacterial diversity and community structure in wastewater treated with different concentrations of ammonia nitrogen using the *Vallisneria natans* ecological interface combined with immobilized microorganisms. The medium-concentration experimental group had the fewest sequencing sequences (43,283), while the high-concentration experimental group had the fewest (42,454). After sequence alignment, operable taxonomic units (OTUs) were identified (similarity > 97%). A total of 2,626 OTUs were obtained from 19 samples in the medium-concentration experiment, with the number of OTUs ranging from 487 to 1,073 per sample, corresponding to SF3a (sample on day 6 of the free bacteria experimental group) and SV1a (sample on day 2 of the *Vallisneria natans* ecological interface), respectively. A total of 2,609 OTUs were obtained from 19 samples in the high-concentration experiment, with the number of OTUs ranging from 538 to 1,272 per sample, corresponding to SCK3b (sample on day 6 of the CK group) and SVC1b (sample on day 2 of the *Vallisneria natans* ecological interface + immobilized bacteria experimental group), respectively. Dilution curves were plotted using the number of randomly selected sequences and the types of OTUs obtained from alignment. It can be seen that the number of OTUs in all samples increases rapidly with the increase of sequencing depth, and then gradually slows down, indicating that the sequencing depth can cover the vast majority of bacterial OTUs.

[0083] Results Analysis: The results are as follows Figure 5 As shown, using the sequence number obtained from the sample with the lowest sequencing count as a benchmark, the same number of sequences were randomly selected from other experimental groups to obtain the number of OTUs for the corresponding samples, which were used to calculate the alpha diversity of each sample in the experimental groups. The Chao index and Shannon index were used to analyze the abundance and diversity of the microbial community in each sample. In the process of treating medium-concentration ammonia nitrogen wastewater by combining the Vallisneria natans ecological interface with immobilized microorganisms, the Chao index of samples from different control groups at different time points was as follows: the largest was SVC1a (Vallisneria natans ecological interface + immobilized bacteria experimental group, day 2 sample), with a value of 1364; the smallest was SF3a (free bacteria experimental group, day 6 sample), with a value of 575. The largest Shannon index was SCa (control group, day 1 sample), with a value of 4.29; the smallest was SV3a (Vallisneria natans group, day 6 sample), with a value of 2.74. As shown in Table 3.

[0084] Table 3 shows the Alpha diversity of water bodies during the ammonia nitrogen concentration wastewater process.

[0085]

[0086]

[0087] In the treatment of high-concentration ammonia nitrogen wastewater using the Vallisneria natans ecological interface combined with immobilized microorganisms, the Chao index of samples from different control groups at different time points was highest for SVC1b (Vallisneria natans ecological interface + immobilized bacteria experimental group, day 2 sample), at 1487, and lowest for SCK3b (control group, day 6 sample), at 630. The Shannon index was highest for SCKb (control group, day 1 sample), at 4.38, and lowest for SVC3b (Vallisneria natans ecological interface + immobilized bacteria experimental group, day 6 sample), at 2.54. (See Table 4).

[0088] Table 4. Alpha diversity of water bodies during high-concentration ammonia nitrogen wastewater treatment.

[0089]

[0090]

[0091] Results Analysis: Comparing the Alpha diversity analysis results of various samples in the experiment, regardless of whether the wastewater contained medium or high concentrations of ammonia nitrogen, the experimental groups containing the Vallisneria natans ecological interface, such as SV (Vallisneria natans ecological interface), SVF (Vallisneria natans ecological interface + free bacteria), and SVC (Vallisneria natans ecological interface + immobilized bacteria), showed significantly higher chao indices than the CK (blank control) group after 7 days of treatment. Conversely, the shannon index decreased, indicating that the presence of Vallisneria natans increased species richness. The experimental groups with added immobilized bacterial agents, such as the SC (immobilized bacteria) group, showed a trend of decreasing chao index and increasing shannon index, indicating that the addition of the bacterial agent increased diversity. In the treatment of medium and high concentrations of nitrogen-polluted water, both the chao and shannon indices generally showed a decreasing trend, consistent with the trend of ammonia nitrogen removal, which is the reason why the relevant functional bacteria were continuously enhanced during the treatment process.

[0092] 2.1.2 Characteristics of bacterial community structure

[0093] like Figure 6As shown, OTUs obtained from all samples were annotated, revealing that the bacteria belonged to 49 phyla and 869 genera. The bacteria were mainly concentrated in 10 phyla: Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Verrucomicrobia, Cyanobacteria, Planctomycetes, Unclassified, Chloroflexi, and Bacteria_Unclassified. The combined content of Proteobacteria, Actinobacteria, Bacteroidetes, and Cyanobacteria exceeded 90.5%, except for SCK3 (84.0%), SC1b (89.0%), and SC3b (89.8%), indicating high stability of the system's bacteria at the phylum level. (2 mg·L⁻¹) -1 In the concentration tests, compared with SCK, the abundance of Proteobacteria in the SC and SF groups remained unchanged, while the abundance of Actinobacteria increased significantly by approximately 15%. In the SV, SVC, and SVF groups, the abundance of Proteobacteria increased significantly by approximately 14.7%–19.6%, while the abundance of Actinobacteria decreased by approximately 4.5%–8.0%. In the 4 mg / L concentration test, compared with SCK, the abundance of Proteobacteria in the SC and SF groups remained essentially unchanged, while the abundance of Proteobacteria in the SV, SVC, and SVF groups increased significantly by approximately 10.6%–22.8%, while the abundance of Bacteroidetes decreased by 7.7%–14.0%. This indicates that the presence of Vallisneria natans increases the abundance of Proteobacteria but decreases the abundance of Actinobacteria. When comparing different concentrations, it was found that at higher ammonia nitrogen concentrations, the abundance of Actinobacteria in SC and SF was significantly reduced, while the abundance of Proteobacteria was increased, and the abundance of Bacteroidetes in SV, SVC, and SVF groups was significantly reduced.

[0094] like Figure 7As shown, at the genus level, species with relatively high abundance include *Comamonadaceae_uncultured*, *Pseudorhodoferax*, *hgcI clade*, *Flavobacterium*, *Enterobacter*, *Nevskia*, *Brevundimonas*, *Sphaerotilus*, *Polynucleobacter*, and *Rhodobacter*. After water treatment, compared to the SCK group, the SV, SVC, and SVF groups showed a significant increase in *Pseudorhodoferax* abundance (14.0%–43.2%), while the SC and SF groups showed a significant increase in *hgcI clade* abundance in the 2 mg / L concentration samples. In the 4 mg / L concentration samples, the SC group showed an increase in *Enterobacter*, while the abundance of *Comamonadaceae_uncultured* decreased to varying degrees in all groups (1.4%–36%). This indicates that the presence of Vallisneria natans increases the abundance of Pseudorhodoferax, while both immobilized inoculants and Vallisneria natans decrease the abundance of Comamonadaceae_uncultured. Comparing different concentrations, at higher concentrations, immobilization or Vallisneria natans increases the abundance of Comamonadaceae_uncultured but decreases the abundance of Flavobacterium.

[0095] 2.1.3 Principal coordinate analysis of differences in bacterial community structure

[0096] like Figure 8 As shown, principal coordinate analysis was performed on the differences in bacterial community structure in samples at different time points during the treatment of water bodies with different concentrations of nitrogen pollution. Significant differences in bacterial community structure were observed between the initial test samples (SCa, SCb) and the experimental groups containing the *Vallisneria natans* ecological interface (SV, SVC, SVF groups) and those without the *Vallisneria natans* ecological interface (SC, SCK, SF groups). These differences were divided into three parts, indicating that the bacterial community structure of each experimental group underwent significant changes compared to the initial samples and during the treatment of nitrogen-polluted water. The significant difference in bacterial community structure between the experimental groups containing and without the *Vallisneria natans* ecological interface indicates that the *Vallisneria natans* ecological interface had a much greater impact on the bacterial community structure changes in the test water than the addition of water-purifying bacteria. Compared to the control group, the bacterial community structure of both the free and immobilized water-purifying bacteria experimental groups changed significantly, with different effects on the overall bacterial community structure.

[0097] 2.2 Diversity analysis of denitrification functional genes nirS

[0098] 2.2.1 Alpha diversity analysis of denitrifying bacterial communities

[0099] like Figure 9 As shown, the denitrification functional gene *nirS* was selected, and high-throughput sequencing was used to analyze the diversity and community structure changes of denitrifying bacteria during the treatment of wastewater with different concentrations of ammonia nitrogen by the *Vallisneria natans* ecological interface combined with immobilized microorganisms. The samples in the medium-concentration experimental group had the fewest sequencing sequences (41,721), while those in the high-concentration experimental group had the fewest (41,559). After sequence alignment, operable taxonomic units (OTUs) were defined (similarity > 97%). A total of 4,241 OTUs were obtained from the 19 samples in the medium-concentration experiment, with the number of OTUs ranging from 438 to 1,720 per sample, corresponding to SF3a (sample on day 6 of the free bacteria experimental group) and SVC1a (sample on day 2 of the *Vallisneria natans* ecological interface + immobilized bacteria experimental group), respectively. A total of 3,780 OTUs were obtained from the 19 samples in the high-concentration experiment, with the number of OTUs ranging from 466 to 1,249 per sample, corresponding to SF2b (sample on day 4 of the free bacteria experimental group) and SVC1b (sample on day 2 of the *Vallisneria natans* ecological interface + immobilized bacteria experimental group), respectively. Dilution curves were plotted using the number of randomly selected sequences and the types of OTUs obtained from their alignment. The results showed that the sequencing depth could cover the vast majority of denitrifying bacterial OTUs.

[0100] Results Analysis: Using the sample with the lowest sequence count as a baseline, the same number of sequences were randomly selected from other experimental groups to obtain the corresponding OTU count, which was used to calculate the Alpha diversity of denitrifying bacteria in each sample of the experimental groups. The Chao and Shannon indices were used to analyze the abundance and diversity of denitrifying microbial communities in each sample. For samples from different experimental groups treated with medium-concentration ammonia nitrogen pollution at different time points, the highest Chao index was 1896 for SVC1a (Vallisneria natans ecological interface + immobilized bacteria experimental group on day 2), and the lowest was 485 for SF3a (free bacteria experimental group on day 6). The highest Shannon index was 5.97 for SCKa (control group on day 1), and the lowest was 3.7 for SC1a (immobilized bacteria group on day 2). See Table 5.

[0101] Table 5 shows the Alpha diversity of denitrifying bacteria in wastewater with high ammonia nitrogen concentrations during the wastewater treatment process.

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[0104] During the treatment of high-concentration ammonia nitrogen wastewater, the highest Chao index and Shannon index for samples from different control groups at different time points were SCKb (sample from the control group on day 1), at 2044 and 6.16 respectively, while the lowest were SF2b (sample from the free bacteria test group on day 4), at 517 and 3.41 respectively. (See Table 6).

[0105] Table 6. Alpha diversity of denitrifying bacteria in high-concentration ammonia nitrogen wastewater.

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[0108] Results Analysis: Comparing the Alpha diversity analysis results of denitrification functional genes in various samples during the experiment, in the treatment of medium-concentration ammonia nitrogen wastewater, after 7 days of experimentation, the chao index of the SVC (Vallisneria natans ecological interface + immobilized bacteria) group was significantly higher than that of the SCK group, while the shannon index was not significantly different, indicating that the Vallisneria natans ecological interface and immobilized bacteria improved the species richness of denitrifying bacteria. In the high-concentration ammonia nitrogen wastewater treatment experiment, the SV (Vallisneria natans ecological interface), SVF (Vallisneria natans ecological interface + free bacteria), and SVC (Vallisneria natans ecological interface + immobilized bacteria) groups with the presence of the Vallisneria natans ecological interface all had significantly higher chao and shannon indices than the SCK (blank control) group, indicating that the Vallisneria natans ecological interface has a significant effect on improving the species richness and diversity of denitrifying microorganisms.

[0109] 2.2.2 Community structure characteristics of denitrifying microorganisms

[0110] Annotation of OTUs obtained from nirS gene sequencing of all samples revealed that denitrifying bacteria belonged to 7 phyla and 150 genera. In both medium and high concentration ammonia nitrogen wastewater treatment groups, over 94.6% of denitrifying bacteria in the groups without the *Vallisneria natans* ecological interface (SV, SVC, SVF, SCK) were concentrated in the phylum Proteobacteria and unclassified bacteria. In the groups with the *Vallisneria natans* ecological interface (SV, SVF, SVC), the proportion of denitrifying bacteria belonging to the phylum Firmicutes and phylum Actinobacteria ranged from 11.9% to 33.3%, significantly higher than in the groups without the *Vallisneria natans* ecological interface, indicating that the application of the *Vallisneria natans* ecological interface improved the species abundance and diversity of denitrifying bacteria. Figure 10 As shown.

[0111] At the genus level, excluding those yet to be classified, denitrifying microorganisms belonging to 10 genera—Arenimonas, Paracoccus, Azoarcus, Rhizobacter, Proteobacteria norank, Exiguobacterium, Kocuria, Dechloromonas, Pseudomonas, and Paucibacter—have relatively high abundances. For example... Figure 11 As shown. Compared with the SCK group, the SVC group showed an increase in the abundance of denitrifying bacteria, primarily belonging to the genera Azoarcus (8.8%–12.4%), Exiguobacterium (5.3%–23.2%), and Kocuria (6.0%–7.0%), while the abundance of denitrifying bacteria was reduced, primarily belonging to the genera Paucibacter (4.0%–9.9%) and Arenimonas (7.0%–12.5%). In the SV, SVF, and SVC experimental groups, where the Vallisneria natans ecological interface was located, the abundance of the genera Azoarcus, Exiguobacterium, and Kocuria significantly increased, while the abundance of the genera Paucibacter significantly decreased. Compared with the SV group, the SVC group showed increased abundances of Rhizobacter and Dechloromonas by 7.8% and 4.1% at medium concentrations, respectively, and increased abundances of Exiguobacterium and Arenimonas by 16.3% and 3.7% at high concentrations, respectively. Comparing the denitrifying communities in the SVC group at different concentrations, high ammonia nitrogen concentrations increased the abundances of Arenimonas, Proteobacteria norank, and Pseudomonas, while Rhizobacter, Dechloromonas, and Unclassified bacteria decreased to varying degrees. In contrast, the abundances of Arenimonas denitrifying bacteria significantly increased (3.0%–9.1%) in the SV, SVC, and SVF groups, while the abundances of Rhizobacter denitrifying bacteria decreased by 11.4%–19.9%. It is speculated that Rhizobacter, Exiguobacterium, Dechloromonas, and Arenimonas played important functional roles in TN removal in the SVC group.

[0112] 2.2.3 Principal coordinate analysis of differences in denitrifying microbial community structure

[0113] Principal coordinate analysis was performed to analyze the differences in denitrifying bacterial community structure in samples taken at different time points during the treatment of water bodies with different concentrations of nitrogen pollution. Figure 12As shown, the denitrifying bacterial community structure differed significantly between the experimental groups containing the *Vallisneria natans* ecological interface (SV, SVC, SVF groups) and those without (SC, SCK, SF groups), indicating that the *Vallisneria natans* ecological interface has a decisive influence on the denitrifying bacterial community during nitrogen-polluted water treatment. Compared to the denitrifying bacterial community structure in the groups without the *Vallisneria natans* ecological interface (SC, SCK, SF groups), which changed significantly over time during treatment, the denitrifying bacterial community structure in the groups containing the *Vallisneria natans* ecological interface remained relatively stable. Significant differences in the denitrifying bacterial community were observed when the *Vallisneria natans* ecological interface was used alone, and when combined with free bacteria or immobilized composite bacteria. This indicates that although the *Vallisneria natans* ecological interface has a decisive influence on the denitrifying bacterial community during nitrogen removal, different application methods of the water-purifying bacteria also affect the changes in the denitrifying bacterial community structure to some extent. This is closely related to the conversion of ammonia nitrogen and the removal of total nitrogen during the treatment process.

[0114] Submerged-plant ecological interface for in-situ treatment of village ponds

[0115] To test the practical application effect of the submerged plant-ecological interface, the *Vallisneria natans*-ecological interface prepared in Example 1 was applied to the ecological restoration of Nanbang Pond in Yangdu Village, Xucun Town, Haining City, Zhejiang Province. Nanbang Pond has a total area of ​​approximately 470 square meters, an average water depth of 85 cm, and water quality classified as below Class V. In addition to installing a propulsion aeration device to maintain dissolved oxygen above 3 mg / L, the project primarily relied on the submerged plant-ecological interface for ecological restoration. The *Vallisneria natans* ecological interface prepared according to Example 1 was laid along the pond bottom, covering a total area of ​​approximately 200 square meters. After the project was implemented in May 2022, samples were collected and monitored on May 22, June 5, and June 31, respectively, over a period of one and a half months, to monitor changes in CODcr, ammonia nitrogen, and total nitrogen in the Nanbang Pond of Yangdu Village, where the ecological restoration project was implemented. The results showed that after about a month and a half, the CODcr, ammonia nitrogen, and total nitrogen in the Nanbang fishpond decreased by 24.2%, 35.2%, and 27.5%, respectively, as shown in Table 7.

[0116] Table 7: Data Tables for In-situ Treatment

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[0119] In summary: 1) Based on the seedling growth results above, it can be seen that the seedling growth of Examples 1-3 is better than that of Example 4, indicating that the addition of magnesium ammonium phosphate helps the seedlings grow healthily and vigorously, and further helps to form a biofilm on the ecological interface, which is beneficial to the subsequent implantation in water level restoration. This is because magnesium ammonium phosphate, also known as struvite, is a slow-release nitrogen and phosphorus fertilizer that can not only meet the growth needs of submerged plants and be implanted into the planting soil of the river, but also will not affect the water body or increase water pollutants.

[0120] 2) Simulated wastewater purification experiments showed that the submerged plant-ecological interface system effectively treated NH4+ in water bodies with ammonia nitrogen concentrations of 2–2.5 mg / L and 4–5 mg / L. + -N removal amount is approximately 46.85–65.15 mg·m³. -2 ·d -1 The TN removal rate was approximately 29.13–41.8 mg·m³. -2 ·d -1 .

[0121] 3) Vallisneria natans ecological interface + immobilized bacterial agent (M5) for 7 days with medium concentration (2-2.5 mg / L) NH4 + -N removal rate was 97.3%, and TN removal rate was 84.2%. For high concentrations (4-5 mg / L) of NH4... + -N removal rate was 82.6%, and TN removal rate was 62.4%. The addition of immobilized microorganisms enhanced TN removal capacity. High-throughput sequencing results of water samples showed that the bacteria belonged to 49 phyla and 869 genera. Proteobacteria, Bacteroidetes, Cyanobacteria, and Actinobacteria were the dominant phyla, with Proteobacteria being the most dominant. The submerged plant-ecological interface system increased the abundance of Proteobacteria and decreased the abundance of Actinobacteria in the simulated wastewater treatment process. Under high ammonia nitrogen concentrations, the abundance of Bacteroidetes in the submerged plant-ecological interface system experimental group was significantly reduced. Denitrifying bacteria belonged to 7 phyla and 150 genera. More than 94.6% of the bacteria possessing the nirS gene were concentrated in Proteobacteria and unclassified bacteria. The submerged plant-ecological interface system experimental group altered the abundance ratio of denitrifying bacteria, increased the abundance of Firmicutes and Actinobacteria as denitrifying bacteria, and decreased the abundance of Proteobacteria and Unclassified denitrifying bacteria. When applied to water bodies, the slow-release microorganisms, whether nitrifying or denitrifying, will form a denitrifying microbial community in the ecological interface system, while using COD-removing microorganisms will help degrade organic matter.

[0122] 4) Existing submerged plants are planted after being pre-cultivated on land, similar to the principle of lawn planting. This is difficult to plant and has high environmental requirements. However, the ecological interface used in this invention is simple to operate in practice. At the same time, since it is a pure natural material, it does not pollute the environment. When planted together with the submerged plants, it slowly degrades and changes over a long period of time, becoming integrated with the substrate without any residue.

[0123] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A submerged plant-ecological interface system, characterized in that, It includes submerged plants, an ecological interface, and a mud mixture; the ecological interface is a blanket-like material processed from wood filaments; the mud mixture contains immobilized microorganisms, which include microorganisms and carriers that encapsulate the microorganisms; the mud mixture also contains magnesium ammonium phosphate.

2. The submerged plant-ecological interface system as described in claim 1, characterized in that, The mud mixture comprises 4.0-5.0 parts water, 4.0-5.0 parts bottom mud, 0.5-1.0 parts magnesium ammonium phosphate, and 0.5-1.0 parts immobilized microorganisms.

3. The submerged plant-ecological interface system as described in claim 2, characterized in that, When used in nitrogen-polluted water bodies, the microorganisms include nitrifying microorganisms and / or denitrifying microorganisms; when used in polluted water bodies with excessive COD, the microorganisms include Bacillus pumilus and / or Enterobacteriaceae.

4. The submerged plant-ecological interface system as described in claim 3, characterized in that, When used in nitrogen-polluted water bodies, the microorganisms include 2.5 parts nitrifying microorganisms and 7.5 parts denitrifying microorganisms, with a total viable count ≥ 10 billion CFU / g; when used in water bodies with excessive COD, the microorganisms include 5.5 parts Bacillus pumilus and 4.5 parts Enterobacteriaceae, with a total viable count ≥ 10 billion CFU / g.

5. The submerged plant-ecological interface system as described in claim 4, characterized in that, The carriers containing microorganisms include sodium alginate, agar, diatomaceous earth, and CaCl2.

6. The submerged plant-ecological interface system as described in claim 5, characterized in that, The submerged plant is Vallisneria seeds; the ecological interface is a coconut fiber mat with a thickness of 1.5-3 cm; the Vallisneria seeds are spread on the surface of the coconut fiber mat; the mud mixture covers the coconut fiber mat and Vallisneria seeds with a thickness of 1-2 mm.

7. The method for preparing the submerged plant-ecological interface system according to any one of claims 1 to 6, characterized in that, Includes the following steps: 1) Soak submerged plants in clean water and cultivate them until they sprout white buds; 2) Soak the ecological interface with clean water, then place the ecological interface in a container to keep it moist. 3) Spread the submerged plants with sprouting white buds on the surface of the ecological interface and cover them with mud mixture, then add the water to be restored or clean water to the thickness of the ecological interface. 4) Once the seedlings have emerged, increase the water level to submerge them until the stems and leaves of the submerged plants grow to more than 5cm and their roots penetrate the ecological interface.

8. A method for in-situ remediation of water bodies, characterized in that, The submerged plant-ecological interface system prepared by the method described in claim 7 is removed from the container and laid on the bottom of the water body to be restored.

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