A method for reducing CH4 emission of constructed wetland, constructed wetland and construction method thereof

Abstract

The application provides a method for reducing CH4 emission of a constructed wetland, a constructed wetland and a construction method thereof, and particularly relates to the field of environmental protection technology.The method for reducing CH4 emission of the constructed wetland comprises the following steps: incorporating biochar loaded nano zero-valent iron into an upper substrate of the constructed wetland; and the incorporation amount of the biochar loaded nano zero-valent iron is 0.5-2% of the mass of the upper substrate.By controlling the content of the biochar loaded nano zero-valent iron, the concentration of soluble organic carbon (DOC) in the constructed wetland is improved, and then the diversity of CH4 oxidizing bacteria and the relative abundance of the genus Methanomassiliicoccus are improved, so that the CH4 oxidation reaction is promoted, and finally the CH4 emission is reduced.Through the data of the examples, it can be known that the average CH4 emission flux of the constructed wetland prepared by the application is 0.22-0.35 mg·C / m 2 / h, which is reduced by 35.6-59.8% compared with the control.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection technology, specifically relating to a method for reducing CH4 emissions from constructed wetlands, constructed wetlands and their construction methods. Background Technology

[0002] Constructed wetlands, a wastewater treatment technology mimicking natural wetland designs, effectively remove agricultural nitrogen pollutants primarily through the interaction between substrates, wetland plants, and microorganisms. Due to their advantages such as low cost, low energy consumption, ease of maintenance, high efficiency, and environmental beautification, they are widely used in the treatment of agricultural non-point source pollution. However, traditional constructed wetlands release a certain amount of greenhouse gases, especially methane (CH4), during operation, causing environmental problems to shift from "water pollution" to "air pollution." CH4 emissions originate from a wide range of human activities and natural processes, with wetland ecosystems constituting the most significant natural emission source, and constructed wetlands accounting for 82% of this. Given the continuous increase in atmospheric CH4 concentration, CH4 has gradually become the second most important anthropogenic greenhouse gas after carbon dioxide (CO2). How to effectively reduce CH4 emissions from constructed wetlands has become a research hotspot for scholars both domestically and internationally. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method for reducing CH4 emissions from constructed wetlands, constructed wetlands, and a method for constructing the same. The method provided by the present invention can effectively reduce CH4 emissions and increase the relative abundance of *Methanobacterium* and *Pleurotrophic Bacteria*.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] This invention provides a method for reducing CH4 emissions from constructed wetlands by incorporating biochar-supported nano-zero valent iron into the upper substrate of the constructed wetland; the amount of biochar-supported nano-zero valent iron incorporated is 0.5 to 2% of the mass of the upper substrate.

[0006] The present invention also provides an artificial wetland, comprising a substrate, the substrate comprising an upper substrate, an intermediate substrate and a lower substrate; the upper substrate comprising first gravel and biochar-supported nano-zero valent iron;

[0007] The biochar-supported nano-zero-valent iron accounts for 0.5 to 2% of the mass of the upper matrix.

[0008] Preferably, the thickness of the upper matrix is ​​16–24 cm.

[0009] Preferably, the particle size of the first gravel is 0.5–1 cm, and the packing density of the first gravel is 1.80–1.90 t / m³. 3 .

[0010] Preferably, the intermediate matrix comprises a second gravel, and the thickness of the intermediate matrix is ​​13-17 cm.

[0011] Preferably, the particle size of the second gravel is 1-2 cm, and the packing density of the second gravel is 1.60-1.80 t / m³. 3 .

[0012] Preferably, the lower matrix comprises quartz sand, and the thickness of the lower matrix is ​​3 to 7 cm.

[0013] Preferably, the particle size of the quartz sand is 0.5–1 cm, and the packing density of the quartz sand is 1.60–1.80 t / m³. 3 .

[0014] Preferably, the mass ratio of nano-zero valent iron to biochar in the biochar-supported nano-zero valent iron is 0.5:1 to 2:1.

[0015] The present invention also provides a method for constructing the artificial wetland described in the above technical solution, comprising the following steps:

[0016] The first gravel and biochar-supported nano-zero-valent iron were mixed to obtain the upper matrix;

[0017] The upper substrate, middle substrate, and lower substrate are filled in sequence to obtain the artificial wetland.

[0018] This invention provides a method for reducing CH4 emissions from constructed wetlands by incorporating biochar-supported nano-zero-valent iron into the upper substrate of the constructed wetland; the amount of biochar-supported nano-zero-valent iron incorporated is 0.5-2% of the mass of the upper substrate. This invention increases the concentration of soluble organic carbon (DOC) in the constructed wetland by controlling the content of biochar-supported nano-zero-valent iron, thereby increasing the diversity of CH4-oxidizing bacteria and the relative abundance of broad-methyltrophic bacteria, thus promoting the CH4 oxidation reaction and ultimately achieving CH4 emission reduction. Data from the examples show that the constructed wetland provided by this invention has an average CH4 emission flux of 0.22-0.35 mg·C / m³. 2 / h, a reduction of 35.6%–59.8% compared to the control. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1This is a schematic diagram of the structure of the artificial wetlands prepared in Examples 1-2 and Comparative Example 1;

[0021] Figure 2 This is a SEM image of the biochar-supported nano-zero-valent iron prepared in this invention.

[0022] Figure 3 The average pH value (a), redox potential (b), and soluble organic carbon concentration (c) of the effluent from the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 are given.

[0023] Figure 4 The dynamic emission flux of CH4 in the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 are a) and the average emission flux of CH4 are b).

[0024] Figure 5 The relative abundance of the CH4 archaea mcrA gene (a) and CH4 oxidizing bacteria pmoA gene (b) in the artificial wetland produced in Examples 1-2 and Comparative Example 1, and the relative abundance ratio of the mcrA gene and the pmoA gene (c) are shown.

[0025] Figure 6 The changes in the genus-level CH4-producing bacterial community structure (a) and the relative abundance of genus-level CH4-producing bacteria (b) in the constructed wetlands prepared in Examples 1-2 and Comparative Example 1;

[0026] Figure 7 The constructed wetland CH4 oxidizing bacteria genus horizontal community structure a) and genus-level CH4 oxidizing bacteria community structure b) obtained in Examples 1-2 and Comparative Example 1;

[0027] Figure 8 Pearson correlation analysis was performed on the average CH4 emission flux, effluent physicochemical properties, CH4-producing bacterial α-diversity index, and relative abundance of CH4-producing bacteria at the genus level of the constructed wetland prepared in Example 1.

[0028] Figure 9 Pearson correlation analysis was performed on the average CH4 emission flux, effluent physicochemical properties, CH4 oxidizing bacteria α diversity index, and genus-level relative abundance of CH4 oxidizing bacteria in the constructed wetland prepared in Example 2. Detailed Implementation

[0029] This invention provides a method for reducing CH4 emissions from constructed wetlands by incorporating biochar-supported nano-zero valent iron into the upper substrate of the constructed wetland; the amount of biochar-supported nano-zero valent iron incorporated is 0.5 to 2% of the mass of the upper substrate.

[0030] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available products well known in the art.

[0031] In this invention, the amount of biochar-supported nano-zero-valent iron incorporated is 0.5% to 2% of the mass of the upper matrix. In specific embodiments, the amount of biochar-supported nano-zero-valent iron incorporated is 0.5%, 1%, 1.5%, or 2% of the mass of the upper matrix. In specific embodiments of this invention, the effect of suppressing CH4 emissions is best when the amount of biochar-supported nano-zero-valent iron incorporated is 1% of the mass of the upper matrix.

[0032] In this invention, the mass ratio of nano-zero valent iron to biochar in the biochar-supported nano-zero valent iron is preferably 0.5:1 to 2:1, and in a specific embodiment it can be 1:1.

[0033] In this invention, the biochar-supported nano-zero-valent iron is preferably prepared using conventional techniques in the art. Specifically, Fe... 2+ The solution and biochar are mixed and sonicated under an inert atmosphere to obtain a mixture; the mixture is then mixed with NaBH4 solution, stirred under an inert atmosphere, and subjected to solid-liquid separation to obtain a solid, which is washed with water and dried to obtain the biochar-supported nano-zero-valent iron. In this invention, the inert atmosphere is preferably nitrogen.

[0034] In this invention, the concentration of the NaBH4 solution is preferably 0.1 to 0.3 mol / L, and the mass ratio of the biochar to the volume of the NaBH4 solution is preferably 1 g: 0.26 to 0.27 L.

[0035] The present invention does not have any special requirements for the solid-liquid separation method; commonly used technical means in the field can be used.

[0036] In this invention, the biochar is preferably prepared using conventional techniques in the art. Specifically, the plant is pulverized and carbonized under an inert atmosphere to obtain biochar. In this invention, the carbonization temperature is preferably 600°C, and the carbonization time is preferably 2 hours.

[0037] In this invention, the plant is preferably reed, and the process of crushing preferably includes washing, cutting, and drying.

[0038] The present invention also provides an artificial wetland, comprising a substrate, the substrate comprising an upper substrate, an intermediate substrate and a lower substrate; the upper substrate comprising first gravel and biochar-supported nano-zero valent iron;

[0039] The biochar-supported nano-zero-valent iron accounts for 0.5 to 2% of the mass of the upper matrix.

[0040] In this invention, the thickness of the upper substrate is preferably 16-24 cm. In specific embodiments, the thickness of the upper substrate can be 16 cm, 18 cm, 20 cm, 22 cm or 24 cm.

[0041] In this invention, the particle size of the first gravel is preferably 0.5–1 cm, and the packing density of the first gravel is preferably 1.80–1.90 t / m³. 3 In a specific embodiment, the filling density of the first gravel can be 1.80 t / m³. 3 1.82t / m 3 1.85t / m 3 1.88t / m 3 Or 1.90t / m 3 .

[0042] In this invention, the composition and preparation method of the biochar-supported nano-zero-valent iron have been discussed above and will not be repeated here.

[0043] In specific embodiments, the mass of the biochar-supported nano-zero-valent iron can account for 0.5%, 1%, 1.5%, or 2% of the mass of the upper matrix. The effect of suppressing CH4 emissions is best when the mass of the biochar-supported nano-zero-valent iron accounts for 1% of the mass of the upper matrix. In this invention, the thickness of the intermediate matrix is ​​preferably 13-17 cm; in specific embodiments, the thickness of the intermediate matrix can be 13 cm, 15 cm, or 17 cm.

[0044] In this invention, the intermediate matrix preferably comprises a second gravel, the particle size of which is preferably 1-2 cm, and the packing density of which is preferably 1.60-1.80 t / m³. 3 In a specific embodiment, the filling density of the second gravel can be 1.60 t / m³. 3 1.65t / m 3 1.70t / m 3 1.75t / m 3 Or 1.80t / m 3 .

[0045] In this invention, the thickness of the lower substrate is preferably 3 to 7 cm. In specific embodiments, the thickness of the lower substrate can be 3 cm, 5 cm or 7 cm.

[0046] In this invention, the lower matrix preferably comprises quartz sand, the particle size of which is preferably 0.5–1 cm, and the filling density of which is preferably 1.60–1.80 t / m³. 3 In a specific embodiment, the filling density of the quartz sand can be 1.60 t / m³. 3 1.65t / m 3 1.70t / m 3 1.75t / m 3 Or 1.80t / m 3 .

[0047] In this invention, the constructed wetland preferably has vents located 42-47 cm from top to bottom. In specific embodiments, the vents can be located at 42 cm, 43 cm, 44 cm, 45 cm, 46 cm, or 47 cm from top to bottom. Providing vents ensures smooth water flow.

[0048] In this invention, the bottom of the constructed wetland preferably includes a water collection area, the thickness of which is preferably 3-8 cm. In an embodiment of this invention, the water collection area is located at the bottom of the polyethylene plastic cylinder.

[0049] In this invention, the constructed wetland preferably also includes emergent plants, which are preferably iris seedlings, and the planting density is preferably 30 plants / m². 2 The present invention preferably involves planting the roots of emergent plants in the intermediate substrate and / or the upper substrate.

[0050] The constructed wetland obtained by this invention increases the concentration of DOC in the constructed wetland by controlling the content of nano-zero valent iron loaded on biochar, thereby increasing the diversity of CH4 oxidizing bacteria and the relative abundance of broad-methyl trophic bacteria, thus promoting the CH4 oxidation reaction and ultimately achieving CH4 emission reduction.

[0051] The present invention also provides a method for constructing the artificial wetland described in the above technical solution, comprising the following steps:

[0052] The first gravel and biochar-supported nano-zero-valent iron were mixed to obtain the upper matrix;

[0053] The upper substrate, middle substrate, and lower substrate are filled in sequence to obtain the artificial wetland.

[0054] The present invention does not have any special requirements for the mixing method; commonly used technical means in the field can be used.

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments. Any modifications, equivalent substitutions, improvements, etc., made to the embodiments of the present invention based on the technical essence and general principles of the present invention without creative effort should be within the protection scope of the present invention.

[0056] Preparation of biochar and biochar-supported nano-zero valent iron

[0057] Reeds, a wetland plant found in the riparian zone of Shanghai, were collected, washed, cut, dried, and pulverized. The collected reeds were then placed in a tubular carbonization furnace, and nitrogen (N2) was introduced for 30 minutes. The temperature was increased from room temperature to 600°C at a rate of 10°C / min and maintained at this temperature for 2 hours to allow for complete pyrolysis. The entire pyrolysis process was carried out under anaerobic conditions. After pyrolysis, the carbonization furnace was allowed to cool to room temperature to obtain biochar.

[0058] Weigh 2.78 g of FeSO4·7H2O and dissolve it in 100 mL of deionized water to prepare 0.1 mol / L Fe. 2+ After the solution was dissolved, it was mixed with 0.56 g of biochar and sonicated under N2 atmosphere for 1 h to obtain a mixture. The mixture was placed under N2 atmosphere, and 150 mL of 0.2 mol / L NaBH4 solution was added dropwise to the mixture using a syringe and stirred for 30 min. The mixture was centrifuged to obtain solid particles, which were washed three times each with deionized water and anhydrous ethanol. The solid particles were then dried by blowing with N2 to obtain biochar-supported nano-zero valent iron, denoted as nZVI-BC, where the mass ratio of nano-zero valent iron to biochar was 1:1.

[0059] The prepared nZVI-BC was scanned by electron microscopy to obtain... Figure 2 ,from Figure 2 As can be seen, nano-zero valent iron is attached to the biochar.

[0060] Construction of artificial wetlands

[0061] Example 1

[0062] A small-scale constructed wetland system was constructed inside a polyethylene plastic cylinder with a diameter of 35.5 cm, a height of 50 cm, and a substrate layer thickness of 40 cm. The constructed wetland has a 5 cm high water collection area at the bottom. To ensure smooth water flow, an air vent is installed 45 cm from the top of the cylinder. Figure 1 As shown.

[0063] The constructed wetland bed substrate consists of three layers: a top layer of 20cm, a middle layer of 15cm, and a bottom layer of 5cm. Gravel (particle size 0.5–1cm; density 1.84t / m³) is used. 3 The mixture of nZVI-BC and nZVI-BC is homogeneous and used as the upper matrix, wherein the mass of nZVI-BC accounts for 1% of the mass of the upper matrix; gravel (particle size 1-2 cm; density 1.70 t / m³) is also used. 3 ) as the intermediate matrix; quartz sand (particle size 0.5-1mm, density 1.70t / m³) 3 (This is used as the lower substrate.) The resulting constructed wetland is as follows: Figure 1 As shown in b).

[0064] Example 2

[0065] The mass of nZVI-BC accounted for 2% of the mass of the upper substrate, and other conditions were the same as in Example 1. The resulting constructed wetland was as follows: Figure 1 As shown in c).

[0066] Comparative Example 1

[0067] The upper matrix contains only gravel (particle size 0.5–1 cm; density 1.84 t / m³). 3 Other conditions are the same as in Example 1. The resulting constructed wetland is as follows: Figure 1 As shown in a).

[0068] Application examples

[0069] The experiment was conducted in a plastic greenhouse at the Zhuangxing Comprehensive Experimental Base of the Shanghai Academy of Agricultural Sciences (30°53′24″N, 121°23′15″E). The greenhouse was open on all sides to avoid the impact of rainfall on the experiment and to maintain the temperature inside the greenhouse consistent with the ambient temperature. The region has a subtropical monsoon climate with an average annual rainfall of 1191.5 mm and an average annual temperature of 16.1℃. Examples 1 and 2 and Comparative Example 1 were each repeated 3 times, for a total of 9 artificial wetlands. On July 2, 2023, uniformly growing iris (Louisiana Iris) seedlings from the experimental station's nursery were transplanted into the artificial wetlands at an initial planting density of 3 seedlings / bucket (30 seedlings / m²). 2 Because nZVI-BC is highly unstable, the constructed wetland system was stabilized for 7 days using river water from the experimental area before the experiment officially began. Based on local farmland runoff discharge conditions, the experimental water was prepared using river water from the experimental area, with an influent TN concentration of 8.51 mg / L and a TOC concentration of 10.88 mg / L.

[0070] Constructed wetland operation, methane gas collection and analysis

[0071] The hydraulic retention time of the constructed wetlands was 7 days, and the average hydraulic loading was 150 mm / d. Simulated runoff was pumped into each constructed wetland weekly from above using a peristaltic pump at a flow rate of 0.43 m³ / d. 3 / h. Water and CH4 gas samples were collected on days 1, 2, 4, and 6 of each hydraulic retention. Physicochemical properties of the constructed wetland effluent, including oxidation-reduction potential (ORP) and pH, were determined on-site using a portable multi-parameter water quality analyzer (HI9829, HAN-NA, Italy). DOC in the effluent was determined using a total organic carbon analyzer (Vario EL III, Elementar, Germany).

[0072] CH4 gas was collected using a static, sealed plexiglass box and a self-developed automatic gas sampling device. The plexiglass box was 50 cm high and 36.5 cm in diameter. During each sampling, the automatic sampling device collected four gas samples from each plexiglass box at 6-minute intervals. Each gas sample was stored in a 1L aluminum foil gas bag (Dalian Delin Gas Packaging Co., Ltd., Dalian, China). The automatic gas sampling device consisted of the following components: a 12V rechargeable battery (NP7-12); a gas pump (FAY4002, 2L / min, Chengdu Qihai Electromechanical Manufacturing Co., Ltd., Chengdu, China); a circuit board box (Nanjing Weina Electronics Co., Ltd., Nanjing, China); and a three-way direct-acting solenoid valve (VDW23-6G-1, SMC Pneumatics Co., Ltd., Tokyo, Japan). The collected samples were sent to the laboratory for CH4 concentration determination using a gas chromatograph (7820A, Agilent Technologies, USA). CH4 standard gas was purchased from the National Center for Standard Materials.

[0073] The formula for calculating CH4 emission flux is:

[0074] F=ρ×V / S×dC / dt×273 / (273+T) Equation 1;

[0075] In Equation 1, F is CH4 (mg) CH4 ·m -2 ·h -1 Emission flux, where ρ is the density of CH4 under standard conditions (kg·m⁻¹). 3 V is the effective volume of the sealed box (m³). 3 S is the area of ​​the base (m²) 2 dC / dt represents the change in CH4 concentration within the sealed chamber per unit time, and T is the average temperature (°C) within the sealed chamber. A linear correlation coefficient R² > 0.90 between the concentration of the four gas samples collected within 18 minutes and time is considered valid.

[0076] The average CH4 emission flux is calculated using the following formula:

[0077]

[0078] In Equation 2, E CH4 CH4 average emission flux (mg) CH4 ·m -2 ·h -1 );F i and F i+1 These represent the CH4 emission fluxes at the i-th and i+1th sampling times, respectively, in mg. CH4 ·m -2 ·h -1 ;t i+1 and t iThe dates are the (i+1)th and ith sampling dates, respectively, in days (d), and n is the total number of measurements within the cumulative emission observation period.

[0079] Determination of methanogenic archaea and methanogenic bacteria

[0080] After the experiment, root matrix samples were collected from five locations close to the plant roots using UV-sterilized forceps and 10mL centrifuge tubes. These samples were thoroughly mixed and stored at -80℃. Guangdong Meggene Technology Co., Ltd. was commissioned to determine the functional genes and community composition of CH4-producing archaea and CH4-oxidizing bacteria. Before the experiment, the gravel samples were placed in beakers, shaken, centrifuged at high speed, and the precipitate was removed for DNA extraction.

[0081] Using extracted DNA from CH4-producing archaea as templates, the mcrA gene sequence of CH4-producing archaea was amplified using forward primer MLf (5′-GGTGGTGTMGGATTCACACARTAYGCWACAGC-3′, SEQ ID NO.1) and reverse primer MLr (5′-TTCATTGCRTAGTTWGGRTAGTT-3′, SEQ ID NO.2). Using extracted DNA from CH4-oxidizing bacteria as templates, the pmoA gene sequence of CH4-oxidizing bacteria was amplified using forward primer pmof1 (5′-GGGGGAACTTCTGGGGITGGAC-3′, SEQ ID NO.3) and reverse primer pmor (5′-GGGGGRCIACGTCITTACCGAA-3′, SEQ ID NO.4). The qPCR program was: 95℃ for 5 min, 40 cycles × (94℃ for 15 s, 60℃ for 15 s), 72℃ for 30 s. The PCR amplification product was purified according to NE. Ultra TM MiSeq libraries were constructed using standard procedures at New England Biolabs, USA, and the constructed amplicon libraries were sequenced using the Illumina Nova 6000 platform with PE250 sequencing. The resulting sequences were clustered with 97% similarity using OTUs (operational taxonomic units).

[0082] Data were analyzed using SPSS 22.0 software. The effects of the physicochemical properties of the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 on the cumulative CH4 emissions were tested using one-way ANOVA and Tukey's HSD (p<0.05). Graphs were generated using Origin 2018 software. Mantel tests were performed and plotted using the ggplot2, vegan, dplyr, linkET, scales, and RColorBrewer packages in R4.3.3.

[0083] Data on the average pH, redox potential, and soluble organic carbon (DOC) concentration of water from eight samples taken from the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 were recorded. Figure 3 .from Figure 3 As shown in a), the addition of nZVI-BC had a significant impact on the pH value of the constructed wetland effluent, with the overall trend being 0% > 1% > 2% (P < 0.05). The addition of nZVI-BC had no significant effect on the redox potential of the constructed wetland effluent. Figure 3 (b) Compared with Comparative Example 1, the DOC concentration in the constructed wetland prepared in Example 1 was significantly increased (P<0.05), while Example 2 had no significant effect on the DOC concentration. Figure 3 c)), wherein the average DOC concentration in Example 1 was 83.4 mg / L.

[0084] The dynamic and average CH4 emission fluxes of the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 were tested to obtain... Figure 4 In Comparative Example 1, the peak CH4 emissions occurred on July 10th (1.33 mg C / m³). 2 / h), July 16 (0.63mg·C / m 2 / h) and July 19 (0.70mg·C / m 2 / h). The peak CH4 emission in Example 1 was on July 14 (0.20 mg·C / m³). 2 / h) and July 19 (0.45mg·C / m 2 / h); In Example 2, the peak emission of CH4 was on July 10 (0.50 mg·C / m³). 2 / h), July 16 (0.33mg·C / m 2 / h) and July 21 (0.60mg·C / m 2 / h)( Figure 4 (a)). Compared with Comparative Example 1, Examples 1-2 both reduced CH4 emission peaks on July 10th and July 16th, with Example 1 showing a more significant reduction (P<0.05). Figure 4(b)). During the entire experimental run, the average CH4 emission flux of Comparative Example 1 was 0.55 mg·C / m³. 2 / h, Example 1: 0.22 mg·C / m 2 / h, Example 2: 0.35 mg·C / m 2 / h. Therefore, compared with Comparative Example 1, Examples 1-2 all significantly reduced the average CH4 emission flux, with the reduction in Example 1 (59.8%) being significantly greater than that in Example 2 (35.6%). Figure 4 (b)

[0085] The relative abundances of the mcrA gene in CH4 archaea and the pmoA gene in CH4 oxidizing bacteria produced in the artificial wetlands prepared in Examples 1-2 and Comparative Example 1 were tested, and the results were obtained. Figure 5 .from Figure 5 As shown in a) and b), the relative abundance of both the mcrA and pmoA genes decreased with increasing nZVI-BC addition. The relative abundance ratio of the mcrA and pmoA genes was calculated to show that... Figure 5 In Example 1, the ratio of mcrA gene to pmoA gene was reduced, while in Example 2, the ratio was increased.

[0086] The Alpha(α) diversity index of CH4 archaea and CH4 oxidizing bacteria produced in the artificial wetland prepared in Examples 1-2 and Comparative Example 1 was tested, and the results are shown in Table 1.

[0087] Table 1. Alpha diversity indices of CH4 archaea and CH4 oxidizing bacteria prepared in artificial wetlands in Examples 1-2 and Comparative Example 1.

[0088]

[0089] Note: Different lowercase letters indicate significant differences between different treatments (P<0.05).

[0090] Compared to Comparative Example 1, Examples 1 and 2 had no significant effect on the α-diversity index of CH4-producing archaea. Unlike CH4-producing archaea, Example 1 significantly improved the α-diversity index of CH4-oxidizing bacteria, especially the Shannon and Simpson indices. Specifically, Example 1 increased the Shannon index of CH4-oxidizing bacteria in the constructed wetland by 52.3% while decreasing the Simpson index by 74.4%, indicating a significant increase in CH4-oxidizing bacteria diversity in Example 1 (a large Shannon index indicates high diversity, and a small Simpson index indicates high diversity). Compared to Comparative Example 1, Example 1 increased the Chao index of CH4-oxidizing bacteria in the constructed wetland by 15.6%, while Example 2 decreased it by 34.3%, indicating that Example 1 increased the abundance of CH4-oxidizing bacteria, while Example 2 decreased their abundance.

[0091] The changes in the structure of CH4-producing archaea communities at the genus level in the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 were investigated using NMDS analysis. Figure 6 (a)). Different constructed wetlands are separated along axis 2 according to the amount of additives. The greater distance between Comparative Example 1 and Examples 1-2 in NMDS space indicates that the genus-level community composition of Comparative Example 1 is different from that of Examples 1-2. The closer distance between Examples 1-2 in NMDS space indicates that they are more similar in genus-level community composition.

[0092] Examples 1-2: CH4-producing archaea community structure at the genus level, as shown below. Figure 6 As shown in b). In Examples 1-2 and Comparative Example 1, the genus *Methanobacterium* had the highest relative abundance, accounting for 65.9% to 89.0%. In Example 1, the relative abundance of *Methanobacterium* increased by 1.76%, while in Example 2, the relative abundance of *Methanobacterium* decreased by 24.7%.

[0093] NMDS analysis of the horizontal community structure of CH4-oxidizing bacteria in constructed wetlands prepared in Examples 1-2 and Comparative Example 1, such as... Figure 7 As shown in a), different constructed wetlands are separated along axis 2 according to the amount of additives. Comparative Example 1 and Example 2 are relatively close in NMDS space, reflecting a certain similarity in their community composition at the genus level. However, Example 1 is significantly farther away from Comparative Example 1 and Example 2 in NMDS space, indicating that its community composition at the genus level differs significantly from the other two.

[0094] The CH4-oxidizing bacterial community structure at the genus level in the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 is as follows: Figure 7As shown in b). In Comparative Example 1 and Example 2, the genus *Methylomonas* had the highest relative abundance, accounting for 89.4% and 99.7%, respectively. In Example 1, the genus *Methylomagnum* had the highest relative abundance, accounting for 55.1%. Therefore, compared with Comparative Example 1, adding the relative abundance of *Methylomonas* from Example 1 increased the relative abundance of *Methylomagnum*.

[0095] The factors affecting CH4 emissions from the constructed wetlands prepared in Examples 1-2 and Comparative Example 1 were analyzed using the Mantel test, yielding the following results: Figures 8-9 .from Figure 8 As can be seen from the data, the average CH4 emission flux is not significantly correlated with the α-diversity index of CH4-producing bacteria, indicating that the average CH4 emission flux is not affected by the diversity and abundance of CH4-producing bacteria.

[0096] The average CH4 emission flux was significantly negatively correlated with the Shannon index of CH4 oxidizing bacteria, and significantly positively correlated with the Simpson index. Figure 9 This indicates that the higher the diversity of CH4-oxidizing bacteria, the lower the CH4 emission. The effluent DOC concentration was significantly positively correlated with the Shannon index of CH4-oxidizing bacteria, and significantly negatively correlated with the Simpson index. Figure 9 This indicates that the diversity of CH4-oxidizing bacteria is more susceptible to the influence of DOC. The average CH4 emission flux was significantly negatively correlated with the relative abundance of the genus *Pseudocytrophicus*. Figure 9 This indicates that *Bacteria spp.* are beneficial for CH4 emission reduction in constructed wetlands. Furthermore, the effluent DOC concentration was significantly positively correlated with the relative abundance of *Bacteria spp.* Figure 9 The results indicate that DOC is a key environmental driver for the genus *Pleurotrophic*. Therefore, the highest DOC concentration was observed in Example 1. This environmental change not only increased the diversity of CH4-oxidizing bacteria but also significantly increased the relative abundance of *Pleurotrophic* among them, thereby accelerating the CH4 oxidation process and ultimately effectively reducing CH4 emissions.

[0097] The constructed wetlands provided by this invention significantly reduce CH4 emissions, with reductions of 59.8% and 35.6% in Examples 1 and 2, respectively. Specifically, the constructed wetland prepared in Example 1 primarily reduces CH4 emissions by increasing the dissolved organic carbon (DOC) content, enhancing the diversity of CH4-oxidizing bacteria, and increasing the relative abundance of broad-methyltrophic bacteria (CH4-oxidizing bacteria), thereby promoting the CH4 oxidation reaction.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for reducing CH4 emissions from constructed wetlands, characterized in that, Biochar-supported nano-zero-valent iron is incorporated into the upper substrate of the constructed wetland; the amount of biochar-supported nano-zero-valent iron incorporated is 0.5-2% of the mass of the upper substrate. The substrate of the constructed wetland includes an upper substrate, an intermediate substrate, and a lower substrate; The upper matrix comprises a first gravel and biochar-supported nano-zero valent iron; The mass ratio of nano-zero valent iron to biochar in the biochar-supported nano-zero valent iron is 0.5:1 to 2:

1.

2. An artificial wetland, characterized in that, The matrix includes an upper matrix, an intermediate matrix, and a lower matrix; the upper matrix includes first gravel and biochar-supported nano-zero valent iron. The biochar-supported nano-zero-valent iron accounts for 0.5-2% of the mass of the upper matrix. The mass ratio of nano-zero valent iron to biochar in the biochar-supported nano-zero valent iron is 0.5:1 to 2:

1.

3. The constructed wetland according to claim 2, characterized in that, The thickness of the upper matrix is ​​16~24cm.

4. The constructed wetland according to claim 2, characterized in that, The first gravel has a particle size of 0.5~1cm and a packing density of 1.80~1.90t / m³. 3 .

5. The constructed wetland according to claim 2, characterized in that, The intermediate matrix includes a second gravel layer, and the thickness of the intermediate matrix is ​​13-17 cm.

6. The constructed wetland according to claim 5, characterized in that, The second gravel has a particle size of 1-2 cm and a packing density of 1.60-1.80 t / m³. 3 .

7. The constructed wetland according to claim 2, characterized in that, The lower matrix comprises quartz sand, and the thickness of the lower matrix is ​​3-7 cm.

8. The constructed wetland according to claim 7, characterized in that, The quartz sand has a particle size of 0.5~1cm and a packing density of 1.60~1.80t / m³. 3 .

9. The method for constructing an artificial wetland according to any one of claims 2 to 8, characterized in that, Includes the following steps: The first gravel and biochar-supported nano-zero-valent iron were mixed to obtain the upper matrix; The upper substrate, middle substrate, and lower substrate are filled in sequence to obtain the artificial wetland.

Images