A subsurface flow constructed wetland and its application in removing antibiotic resistance genes from aquaculture water.

Abstract

This invention relates to the field of ecological protection technology, and more particularly to a subsurface flow constructed wetland and its application in removing antibiotic resistance genes (ARGs) from aquaculture water. The constructed wetland includes a water purification zone; the water purification zone is situated above the bottom sediment and comprises, in sequence, a sand and gravel layer, a gravel layer, a volcanic rock layer, and water; the thickness of the sand and gravel layer is 0.2–0.5 m; the thickness of the gravel layer is 0.1–0.5 m; and the thickness of the volcanic rock layer is 0.1–0.5 m. Antibiotic resistance genes are a new type of environmental pollutant. This invention utilizes a specific subsurface flow constructed wetland constructed from sand and gravel layers and volcanic rock layers. Passing aquaculture water through this subsurface flow constructed wetland before discharge can effectively remove the content of antibiotic resistance genes in the aquaculture water, which is of great significance in the technical field of removing antibiotic resistance genes from aquaculture water.

Description

Technical Field

[0001] This invention relates to the field of ecological protection technology, and in particular to a subsurface flow constructed wetland and its application in removing antibiotic resistance genes from aquaculture water. Background Technology

[0002] Inland freshwater pond aquaculture is one of the main aquaculture methods. With the development of the aquaculture industry, antibiotics are used extensively as aquaculture drugs or in mixed feeds, promoting the development of resistant bacteria and antibiotic resistance genes (ARGs) in the aquaculture environment and within cultured organisms. In the aquaculture environment, resistance genes can be horizontally transferred between different microbial populations via mobile genetic elements, directly entering cultured organisms and ultimately undergoing horizontal gene transfer through the food chain and the human gut microbiota. This makes the aquaculture environment a potential reservoir for antibiotics and ARGs. Currently, ARGs have been reported to be detected in various environmental media (water, soil, sediment) in aquaculture, indicating that aquaculture farms have become reservoirs of ARGs in the environment.

[0003] Antibiotics are widely used in current technologies, providing effective treatments for various infectious diseases. However, with the increasing use of antibiotics, the number of drug-resistant bacteria carrying antibiotic resistance genes is also increasing. Due to the high stability and low hydrophilicity of antibiotics, they are difficult to degrade naturally in the aquatic environment, leading to the formation of a large number of drug-resistant microorganisms and transferable antibiotic resistance genes. Studies have detected dozens of classes and thousands of subtypes of antibiotic resistance genes in ecological recirculating aquaculture systems. Pond sediment is a major location for antibiotic resistance genes, containing a large number of pollutants (such as ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen) and microorganisms, which provide conditions for the persistence of antibiotic resistance genes in the ecosystem and their spread into water bodies. Existing water treatment technologies can effectively remove substances such as COD, ammonia nitrogen, total nitrogen, and total phosphorus from water bodies, but their effectiveness in removing emerging pollutants such as antibiotic resistance genes is minimal.

[0004] Existing technologies include various methods for removing antibiotic resistance genes from polluted water, such as membrane bioreactors (MBRs) (Chinese patent CN106698652A), which is considered an effective technology for removing antibiotic resistance genes from wastewater. Large numbers of pathogens and resistance genes can be detected in the wastewater retained by the MBR membrane. However, this method is costly and energy-intensive, and its maintenance and management are more complex. Another method is the removal of antibiotic resistance genes using a vacuum ultraviolet / potassium persulfate coupled advanced oxidation process (Chinese patent CN115321637A), which is applicable to the removal of different types of antibiotic resistance genes. However, it also suffers from numerous processes and complex equipment. Other methods include adsorption and electrolysis for removing antibiotic resistance genes, but these methods are costly and cumbersome, making them unsuitable for aquaculture. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a subsurface flow constructed wetland and its application in removing antibiotic resistance genes from aquaculture water. By constructing a specific constructed wetland, the content of antibiotic resistance genes is significantly reduced when wastewater or aquaculture water rich in antibiotic resistance genes passes through it.

[0006] Existing technologies often employ complex and multi-step methods such as membrane bioreactors, vacuum ultraviolet light, or advanced oxidation to remove antibiotic resistance genes from water bodies. However, the high cost of use and maintenance makes these methods difficult to promote. This invention, through research, presents a subsurface flow constructed wetland that can quickly and effectively remove antibiotic resistance genes from aquaculture water bodies. Prior to this, there was no technical means to apply constructed wetlands to the removal of antibiotic resistance genes from aquaculture water bodies.

[0007] In a first aspect, the present invention provides a subsurface flow artificial wetland for purifying aquaculture water, comprising a water purification zone; the water purification zone comprises, in sequence, a sand and gravel layer, a gravel layer, a volcanic rock layer, and water.

[0008] The thickness of the sand and gravel layer is 0.2–0.5 m; the thickness of the gravel layer is 0.1–0.5 m; and the thickness of the volcanic rock layer is 0.1–0.5 m.

[0009] Furthermore, the particle size of the sand and gravel in the sand and gravel layer is 0.15–5 mm; the particle size of the gravel in the gravel layer is 3–8 cm; and the particle size of the volcanic rock in the volcanic rock layer is 3–15 mm.

[0010] In the subsurface flow constructed wetland provided by this invention, the sand and gravel layer protects the bottom environment and prevents excessive soil from being stirred up by the water flow. The gravel layer filters nitrogen from the water and serves as a substrate for algae, forming a relatively stable ecological environment. The volcanic rock layer adsorbs pollutants such as odors and particulate matter in the aquaculture water. More importantly, after constructing the constructed wetland in this way, a stable and specific ecological environment can be formed. This ecological environment can significantly reduce antibiotic resistance genes in the aquaculture water through various means. For example, the combined action of various microorganisms in the specific ecological environment can effectively remove these antibiotic resistance genes. Ultimately, after multi-layered purification by the subsurface flow constructed wetland, the level of antibiotic resistance genes in the water is significantly reduced.

[0011] Furthermore, the subsurface flow constructed wetland also includes a cattail area; the cattail area and the water purification area are connected by a pipe; at one end of the water purification area, the pipe opening is located in the sand and gravel layer.

[0012] Furthermore, cattails are present at the water inlet of the cattail area, with a density of 1-3 plants / m². 2 .

[0013] Furthermore, the depth of the subsurface flow constructed wetland is 1 to 3 meters.

[0014] Cattails can play a role in the initial purification of aquaculture water. They can reduce the content of phosphorus and nitrogen in the aquaculture water, and can also play a role in metabolism and degradation to a certain extent, reducing the content of microorganisms containing antibiotic resistance genes in the water.

[0015] Secondly, the present invention provides the application of the aforementioned subsurface flow constructed wetland in the removal of antibiotic resistance genes from water bodies.

[0016] Furthermore, the application includes: the water body enters the subsurface flow constructed wetland through the reed area; it enters the water purification area through pipes, and then passes through the sand and gravel layer, the gravel layer and the volcanic rock layer in sequence before being discharged.

[0017] Furthermore, the water body includes one or more of the following: domestic sewage, industrial wastewater, or aquaculture water.

[0018] Furthermore, the water body in question is an aquaculture water body.

[0019] Furthermore, the aquaculture water body is an aquaculture water body that has undergone three-stage ecological ditch treatment.

[0020] The present invention has the following beneficial effects:

[0021] This invention provides a subsurface flow constructed wetland, which comprises, in sequence, a sand and gravel layer, a gravel layer, and a volcanic rock layer above the bottom sediment. The invention's research has found that passing aquaculture water through this subsurface flow constructed wetland can significantly reduce the level of antibiotic resistance genes in the water. Previously, there was no technical means to apply constructed wetlands to remove antibiotic resistance genes from aquaculture water. The subsurface flow constructed wetland provided by this invention, which can be used to remove antibiotic resistance genes from polluted water, is of great significance in the technical field of water purification and reducing the total content of antibiotic resistance genes in the environment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of a subsurface flow constructed wetland provided in Embodiment 1 of the present invention.

[0024] Figure 2 This is a schematic diagram of a subsurface flow constructed wetland with a cattail area provided in Embodiment 1 of the present invention.

[0025] Figure 3 This is a schematic diagram showing the changes in the distribution characteristics of antibiotic resistance genes in aquaculture water before and after passing through a subsurface flow constructed wetland, as provided in Embodiment 2 of the present invention; where A is the inlet and B is the outlet.

[0026] Figure 4 This is the removal effect of sulfonamide and tetracycline antibiotic resistance genes in aquaculture water before and after passing through a subsurface flow constructed wetland, as provided in Example 2 of the present invention; the left figure shows the comparison of the copy numbers of total sulfonamide antibiotic resistance genes sul, sul1 and sul2 at the inlet and outlet, and the right figure shows the comparison of the copy numbers of total tetracycline antibiotic resistance genes tet, tetg and tetx at the inlet and outlet.

[0027] Figure 5 These are the microbial amplitude and antibiotic resistance genes in the subsurface flow constructed wetland provided in Embodiment 3 of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0029] Example 1

[0030] This embodiment provides a subsurface flow constructed wetland, such as Figure 1 As shown, Figure 1 The purification water body area in the subsurface flow constructed wetland consists of a sand and gravel layer 2, a gravel layer 3, a volcanic rock layer 4, and a water body 5, which are arranged in sequence above the bottom sediment 1.

[0031] In practical applications, the size of the water purification area can be adjusted according to the actual situation, especially the length and width. In this embodiment, the area is 10 meters long, 4 meters wide, and 1 meter high.

[0032] Above the bottom sediment 1 are layers of sand and gravel 2, gravel 3, and volcanic rock 4. In this embodiment, the thickness of each layer is 0.25 meters. The sand and gravel layer 2 is mainly used to protect the bottom environment of the pond and prevent the water flow from stirring up too much mud. It also provides a preliminary filtration effect on the water. The gravel layer 3 can filter nitrogen from the water and can also serve as an attachment substrate for algae, forming a stable microbial barrier and thus further removing antibiotic resistance genes. The volcanic rock layer 4 can adsorb pollutants such as odors and particulate matter in the aquaculture water.

[0033] During use, the aquaculture water is introduced into the purified water area from the sand and gravel layer 2, and then passes through the sand and gravel layer 2, the gravel layer 3 and the volcanic rock layer 4 to reach the water body 5, and then discharged.

[0034] Furthermore, the subsurface flow constructed wetland provided in this embodiment also includes a cattail area, such as... Figure 2 As shown, the cattail area and the purified water area are connected by a pipe 7, with one end of the pipe located at the bottom of the cattail area and the other end located in the sand and gravel layer 2 of the purified water area.

[0035] Cattails are planted on the upper part of the water body in the cattail area at a density of 2 plants / m². 2 When in use, the aquaculture water enters from the cattail area, passes through the pipes at the bottom of the cattail area, enters the purified water area, and then passes through the sand and gravel layer 2, the gravel layer 3, and the volcanic rock layer 4 in sequence to reach the water body 5, and is then discharged.

[0036] Example 2

[0037] This embodiment provides an antibiotic resistance gene removal assay, and the specific test method is as follows:

[0038] 1. Using the subsurface flow constructed wetland provided in Example 1 as the experimental object, it is 20m long, 4m wide, and 1m deep. Cattails are planted at the inlet of the cattail area at a density of 2 plants / m². 2 In the water purification area, volcanic rock layer, gravel layer and sand and gravel layer are laid in sequence on the bottom mud, each layer is 0.25 meters thick.

[0039] 2. Aquaculture wastewater is introduced from the reed bed area and purified through bottom pipes. Water samples are collected from the inlet and outlet of the subsurface flow constructed wetland. NH4 levels are determined according to national standard methods. + -N, NO2 - -N, NO3 - Physicochemical indicators of water quality such as nitrogen, total phosphorus, total nitrogen, and carbon dioxide (TP, TN, and CO

[0040] 4. The above process yields the following results:

[0041] The water quality test results are shown in Table 1:

[0042] Table 1 Water Quality Statistics Results

[0043] Conventional water quality indicators T DO SAL EC TDS pH Inlet water Inf 24.83 6.31 0.22 557 365.7 7.7 Eff of water output 22.77 5.76 0.25 588.3 381.7 7.28 Pollutant water quality indicators TN <![CDATA[NO2-N]]> <![CDATA[NO3-N]]> <![CDATA[NH3-N]]> TP COD Inlet water Inf 5.27 0.21 1.85 0.034 1.2 51.7 Eff of water output 1.88 0.12 0.97 0.017 0.84 27.3 Removal efficiency (%) 64.24 42.19 47.57 49.51 29.72 47.1

[0044] The distribution results of antibiotic resistance genes are as follows: Figure 3 As shown, the specific removal effect is as follows: Figure 4 As shown in the results, after purification by the subsurface flow constructed wetland used in this invention, the total amount of antibiotic resistance genes in the aquaculture water significantly decreased, with the total SUL copy number decreasing from approximately 530 to less than 50; and the total TET copy number decreasing from approximately 70 to approximately 40. Therefore, it can be concluded that when the aquaculture water is treated by the subsurface flow constructed wetland obtained by this invention, the proportion of antibiotic resistance genes significantly decreases.

[0045] Example 3

[0046] To further highlight the mechanism by which this invention removes antibiotic resistance genes, the abundance of microorganisms in subsurface flow constructed wetlands was detected using the following methods:

[0047] 1. Microorganisms were enriched by filtration using a 0.45μm filter membrane and stored in a -80℃ refrigerator.

[0048] 2. DNA and metagenomics were extracted from the enriched microorganisms using the following methods:

[0049] (1) Sample DNA extraction and metagenomic sequencing

[0050] Total genomic DNA was extracted using the Power Water DNA Isolation Kit (MOBIO, USA). Metagenomics techniques were employed for sample sequencing. Multiple parallel reads were sequenced using Illumina HiSeq, and after quality control, the reads were assembled using IDBA-UD (https: / / i.cs.hku.hk / ~alse / hkubrg / projrcts / idba_ud / ). Gene prediction was performed using MetaGene (http: / / metagene.cb.ku-tokyo.ac.jp / ) based on the assembly results.

[0051] (2) Bioinformatics Analysis and Data Analysis

[0052] A non-redundant gene set was constructed and compared with the ARDB (antibiotic resistance gene database) using the "strict" alignment parameter to obtain ARG annotation results. Correlation and difference analyses were performed using R software; p < 0.05 was considered statistically significant.

[0053] The results of this invention's detection of microbial abundance in the sand, gravel, and volcanic rock layers of subsurface flow constructed wetlands show that each layer contains a variety of microorganisms. Furthermore, from... Figure 5 The results showed that Bacteroidetes, Cyanobacteria, and Chloroflexi were significantly correlated with the removal of most antibiotic resistance genes (P<0.01). The presence of these microorganisms may be the reason why the subsurface flow constructed wetland provided by this invention can effectively reduce antibiotic resistance genes in aquaculture water.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a subsurface flow constructed wetland for purifying aquaculture water in removing antibiotic resistance genes from water, characterized in that, The subsurface flow constructed wetland includes a water purification zone and a cattail zone; The water purification zone consists of a sand and gravel layer, a gravel layer, a volcanic rock layer, and water, in sequence above the bottom sediment. The thickness of the sand and gravel layer is 0.2~0.5m; the thickness of the gravel layer is 0.1~0.5m; the thickness of the volcanic rock layer is 0.1~0.5m; The layers of the subsurface flow constructed wetland include Bacteroidetes, Cyanobacteria, and Chloroflexi. Cattails are present at the water inlet of the cattail area; the cattail area and the water purification area are connected by a pipe; at one end of the water purification area, the pipe opening is located in the sand and gravel layer.

2. The application according to claim 1, characterized in that, The sand and gravel in the sand and gravel layer has a particle size of 0.15~5mm; and / or, the gravel in the gravel layer has a particle size of 3~8cm; and / or, the volcanic rock in the volcanic rock layer has a particle size of 3~15mm.

3. The application according to claim 1, characterized in that, The density of the cattails is 1-3 plants / m². 2 .

4. The application according to any one of claims 1-3, characterized in that, The depth of the subsurface flow constructed wetland is 1 to 3 meters.

5. The application according to claim 1, characterized in that, The application includes: the water body enters the subsurface flow constructed wetland through the cattail area; it enters the water purification area through pipes, and then passes through the sand and gravel layer, the gravel layer and the volcanic rock layer in sequence before being discharged.

6. The application according to claim 1 or 5, characterized in that, The water body includes one or more of the following: domestic sewage, industrial wastewater, or aquaculture water.

7. The application according to claim 6, characterized in that, The water body in question is an aquaculture water body.

8. The application according to claim 7, characterized in that, The aquaculture water body refers to the tailwater from the aquaculture pond.

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