Method for improving denitrification efficiency of riparian wetland and application thereof

Abstract

The application provides a method for improving the denitrification efficiency of a riparian wetland and application, and belongs to the technical field of environmental engineering. The method for improving the denitrification efficiency of the riparian wetland can effectively improve the removal efficiency of total nitrogen (TN) in surface runoff water of the riparian wetland, prevent nitrogen from polluting groundwater through leaching and runoff loss, and cause water body eutrophication. Moreover, compared with other types of biochar (fresh biochar and soil aged biochar), the method for improving the denitrification efficiency of the riparian wetland uses greenhouse aged biochar as the material, which not only obtains better treatment effect in reducing TN, NO3 ‑ -N and / or NH4 + -N leaching in the runoff water, but also significantly improves the nitrogen cycle functional gene copy number in the riparian wetland soil in the biological factor aspect.

Description

Technical Field

[0001] This invention belongs to the field of environmental engineering technology, specifically relating to a method and application for improving nitrogen removal efficiency in riparian wetlands. Background Technology

[0002] Improper and excessive application of chemical fertilizers in agriculture leads to the rapid loss of nitrogen through ammonia volatilization, nitrogenous gases, leaching, and runoff. Leaching and runoff account for nearly 30% of fertilizer application. The large amounts of nitrogen lost through leaching and runoff pollute groundwater and cause eutrophication, posing a threat to human health. Therefore, it is urgent to develop effective strategies to reduce nitrogen leaching and runoff loss into water bodies.

[0003] Riparian zones, located at the boundary between terrestrial and aquatic ecosystems, have been shown to prevent nitrogen load from entering freshwater areas. Mayer et al., in a 2007 meta-analysis of 88 studies, reported an average nitrogen removal efficiency of 67.5% for riparian zones (see: Mayer, PM, Reynolds Jr., SK, McCutchen, MD, Canfield, TJ, 2007. Meta-Analysis of Nitrogen Removal in Riparian Buffers. Journal of Environmental Quality 36(4), 1172-1180.). However, riparian zones do not always act as nitrogen buffers. They can also be sources of nitrogen pollution in water bodies (see: Inamdar, S., 2007. Exports of dissolved ammonium (NH4+)). + (during storm events across multiple catchments in a glaciated forested watershed. Environmental Monitoring and Assessment 133(1), 347-363.). Therefore, new methods are needed to improve nitrogen removal efficiency in riparian zones. Summary of the Invention

[0004] The purpose of this invention is to provide a method and its application for improving nitrogen removal efficiency in riparian wetlands. The method described in this invention can effectively improve the removal efficiency of nitrogen (TN) from surface runoff water, preventing nitrogen from polluting groundwater through leaching and runoff, thus preventing eutrophication and posing a threat to human health.

[0005] This invention provides a method for improving nitrogen removal efficiency in riparian wetlands, comprising the following steps:

[0006] 20% to 40% by volume of greenhouse-aged biochar was mixed into the soil in the riverbank zone to obtain a soil-biochar mixture;

[0007] The topsoil of the riparian zone was replaced with a soil-biochar mixture, wherein the thickness of the topsoil of the riparian zone was ≤40cm.

[0008] Preferably, compaction is performed after replacing the topsoil of the riverbank.

[0009] Preferably, the biochar has a particle size of <2 mm.

[0010] Preferably, the greenhouse-aged biochar is obtained through the following steps:

[0011] Fresh biochar is placed in a greenhouse and exposed to natural sunlight for 2 to 4 consecutive years.

[0012] Preferably, after replacing the topsoil of the riverbank with a soil-biochar mixture, quartz sand is laid on the surface of the soil-biochar mixture.

[0013] Preferably, the thickness of the paving is 4 to 6 cm.

[0014] This invention also provides the method described above for improving the denitrification efficiency of riparian wetlands in reducing TN and NO3 in runoff water. - -N and / or NH4 + Applications of -N leaching.

[0015] The present invention also provides the application of the method for improving nitrogen removal efficiency in riparian wetlands described above in increasing the content of nitrogen cycling functional genes in riparian soils.

[0016] Preferably, the nitrogen cycle functional genes include nasA, nirK2, and / or nosZ2.

[0017] The present invention also provides the application of the method for improving the denitrification efficiency of riparian wetlands described in the above technical solution in enhancing the assimilation nitrate reduction rate and / or denitrification rate.

[0018] This invention provides a method for improving the nitrogen removal efficiency of riparian wetlands, effectively enhancing the removal efficiency of total nitrogen (TN) from surface runoff and preventing nitrogen from polluting groundwater and causing eutrophication through leaching and runoff. Furthermore, compared to other types of biochar (fresh biochar and soil-aged biochar), the method described in this invention uses greenhouse-aged biochar as the material, which not only reduces TN and NO in runoff water... 3- -N and / or NH4 +The method achieved better treatment results in nitrogen leaching and significantly increased the copy number of nitrogen cycling functional genes in riparian wetland soils from a biological perspective. In particular, the nasA gene, involved in nitrate reduction assimilation, and the key genes nirK2 and nosZ2 in denitrification both reached their maximum copy number using the method described in this invention for improving nitrogen removal efficiency in riparian wetlands. This demonstrates that the method described in this invention, using greenhouse-aged biochar, significantly enhances the rates of nitrate reduction and denitrification compared to using other biochars, thereby further accelerating NO reduction. 3- -N and NO 2- The conversion process of -N. 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 1 This is a schematic diagram of the leaching test structure provided by the present invention;

[0021] Figure 2 The different treatment groups provided by this invention for influent and effluent TN and NO 3- -N, NH4 + -N and NO 2- -N concentration variation graph;

[0022] Figure 3 This is a comparison chart of TN removal efficiency for different treatment groups provided by the present invention; different lowercase letters indicate significant differences between different treatments (P<0.05);

[0023] Figure 4 Fourier transform infrared spectra of different treatment groups before and after leaching tests provided by the present invention; wherein, (a) is the fresh biochar treatment group; (b) is the greenhouse aged biochar treatment group; and (c) is the soil aged biochar treatment group.

[0024] Figure 5 This is a comparison diagram of soil gene copy numbers in different treatment groups provided by the present invention; where different lowercase letters indicate significant differences between different treatments (P<0.05);

[0025] Figure 6 Pearson correlation analysis diagram of nitrogen cycle functional genes and TN removal efficiency provided by the present invention;

[0026] Figure 7This is a multiple regression tree diagram illustrating the influence of soil characteristics on nitrogen cycle genes, provided by the present invention. Detailed Implementation

[0027] This invention provides a method for improving nitrogen removal efficiency in riparian wetlands, comprising the following steps: mixing 20% ​​to 40% by volume of greenhouse-aged biochar into the riparian soil to obtain a soil-biochar mixture; replacing the riparian surface soil with the soil-biochar mixture, wherein the thickness of the riparian surface soil is ≤40cm.

[0028] In a specific embodiment, the method for improving the nitrogen removal efficiency of riparian wetlands can effectively improve the removal efficiency of total nitrogen from surface runoff water, preventing nitrogen from polluting groundwater and causing eutrophication through leaching and runoff. In a specific embodiment, after replacing the riparian topsoil, compaction is performed. In a specific embodiment, the compaction degree is such that the original bulk density of the riparian soil is restored. In a specific embodiment, the particle size of the greenhouse-aged biochar is <2mm.

[0029] In a specific embodiment, the greenhouse-aged biochar is obtained through the following steps:

[0030] Fresh biochar is placed in a greenhouse and exposed to natural sunlight for 2 to 4 consecutive years.

[0031] In a specific embodiment, the fresh biochar can be made from wood at a pyrolysis temperature of 550°C. Specifically, the wood is collected, washed, cut, dried, and placed in a carbonization furnace. Nitrogen gas is introduced for 1 hour, and then the temperature is raised from room temperature to 550°C at a rate of 15°C / min. This temperature is then maintained at the highest temperature for 2 hours to obtain fresh biochar. In a specific embodiment, the greenhouse can be a glass greenhouse or a fully transparent glass greenhouse. In a specific embodiment, the fully transparent glass greenhouse can have a solar intensity of 800–1000 watts / square meter on sunny days in summer and 300–500 watts / square meter on sunny days in winter, with an average annual total solar radiation of 1100–1400 kilowatt-hours / square meter.

[0032] In a specific embodiment, after replacing the topsoil of the riverbank with a soil-biochar mixture, silica sand is laid on the surface of the soil-biochar mixture. In this embodiment, laying silica sand helps prevent the soil / soil-biochar mixture from being washed away. In this embodiment, the thickness of the quartz sand layer is 4–6 cm.

[0033] This invention also provides the method described above for improving the denitrification efficiency of riparian wetlands in reducing TN and NO in runoff water. 3- -N and / or NH4 + Applications of -N leaching.

[0034] This invention also provides the application of the method for improving nitrogen removal efficiency in riparian wetlands described above in increasing the content of nitrogen cycling functional genes in riparian soils. In a specific embodiment, the nitrogen cycling functional genes include nasA, nirK2, and / or nosZ2. In a specific embodiment, nasA is a gene that promotes the assimilation of nitrate reduction. In a specific embodiment, nirK2 and nosZ2 are denitrification genes.

[0035] The present invention also provides the application of the method for improving the denitrification efficiency of riparian wetlands described in the above technical solution in enhancing the assimilation nitrate reduction rate and / or denitrification rate.

[0036] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method and application for improving nitrogen removal efficiency in riparian wetlands, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Some experimental materials and preparation methods.

[0039] 1. Soil.

[0040] Soil samples were collected from the riparian zone of the Zhuangxing Comprehensive Experimental Station, Shanghai Academy of Agricultural Sciences (30°53′N, 121°23′E). This area has a subtropical monsoon climate with an average annual precipitation of 1191.5 mm, an evaporation rate of 1236.8 mm, and an average annual temperature of 16.1℃. The annual sunshine duration is 1900.2 h, and the frost-free period is 224.4 days. The top 0–20 cm soil samples from the riparian zone contained total organic carbon (SOC), total nitrogen (TN), and ammonium nitrogen (NH4+). + -N), nitrate nitrogen (NO) 3- -N), nitrite nitrogen (NO) 2- The available phosphorus (AP), cation exchange capacity (CEC), and pH were 13.5 g / kg. -1 0.24 g·kg -1 5.45 mg·kg -1 24.1 mg·kg -1 0.94 mg·kg -1 11.6 mg·kg -1 19.7 cmol·kg -1 And 7.44.

[0041] 2. Biochar.

[0042] After collecting, washing, cutting, and drying the wood, it was placed in a carbonization furnace and purged with nitrogen for 1 hour. The temperature was then increased from room temperature to 550°C at a rate of 15°C / min and maintained at the highest temperature for 2 hours to obtain fresh biochar (FBC). Some of the fresh biochar obtained was divided into two parts: one part was buried in riverbank soil for natural aging in the soil environment to obtain soil-aged biochar (SBC); the other part was placed in a greenhouse for natural aging under sunlight to obtain greenhouse-aged biochar (GBC). The specific preparation process is as follows:

[0043] Soil aging biochar: A 30cm layer of topsoil was removed from the original riparian zone. A 10cm layer of riparian soil + fresh biochar mixture (riparian soil:fresh biochar ratio 1:1) was then added at a depth of 20-30cm. After filling, riparian soil was used to cover the top 0-20cm layer, and turf was planted. After 36 months, the biochar added to the riparian zone was collected, and visible biochar particles were separated from the soil sample using tweezers. The hand-selected biochar particles were then suspended in distilled water at a 1:10 (w / v) ratio, and the solution was gently shaken to remove any adhering soil particles. The biochar was then washed four times with distilled water; the washing process should not affect the properties of the biochar. Finally, the washed biochar particles were dried in a convection oven at 60°C to obtain soil aging biochar.

[0044] Greenhouse-aged biochar: Fresh biochar is placed in a fully transparent glass greenhouse and exposed to natural light for three consecutive years to produce greenhouse-aged biochar.

[0045] The functional groups of FBC, SBC, and GBC were determined by Fourier transform infrared spectroscopy. The BET surface area, pH value, and elemental composition of FBC, SBC, and GBC were also determined. The concentrations of TN and NH4+ in FBC, SBC, and GBC were also analyzed. + -N, NO3 - -N, NO2 --N, AP, CEC, and pH were determined according to the standard method described in Bi (2019) (see: Bi, Y., Cai, S., Wang, Y., Xia, Y., Zhao, X., Wang, S., Xing, G., 2019. Assessing the viability of soil successive strawbiochar amendment based on a five-year column trial with six different soils: Views from crop production, carbon sequestration and net ecosystem economic benefits. J Environ Manage 245, 173-186.).

[0046] Example 2

[0047] Leaching test.

[0048] 1. Experimental procedures.

[0049] The soil and biochar prepared in Example 1 were used to conduct leaching tests in specially designed polyvinyl chloride (PVC) columns, each 55 cm high and 12 cm in inner diameter. A schematic diagram of the leaching test structure is shown below. Figure 1 As shown.

[0050] The experiment included four treatments:

[0051] (1) No biochar applied (control);

[0052] (2) Apply fresh biochar (FBC);

[0053] (3) Apply greenhouse aged biochar (GBC);

[0054] (4) Apply soil aging biochar (SBC).

[0055] Each treatment was repeated three times. First, air-dried soil (0-20 cm) with a thickness of <2 mm was manually mixed with fresh biochar, greenhouse-aged biochar, or soil-aged biochar (<2 mm) sieved at 20% (v / v) to obtain soil-biochar mixtures.

[0056] Before filling the PVC column with the soil / soil-biochar mixture, attach a 500-mesh nylon cloth to the bottom of the column, and place a 5cm thick layer of quartz sand on the nylon cloth to prevent the soil / soil-biochar mixture from being washed away. Then, first lay a 20cm thick layer of air-dried soil (particle size <2mm) on the quartz sand, followed by a 20cm thick layer of soil-biochar mixture. Mix the mixture several times by gently shaking and turning it, based on the original bulk density of each layer in the undisturbed riverbank soil, and then compact it with a wooden mallet to ensure consistent density within and between columns. Finally, place a 5cm thick layer of quartz sand on top of the soil column to ensure even moisture distribution.

[0057] Before starting the leaching experiment, deionized water was added to all soil columns until the water level reached 60% of field capacity. The soil columns were then pre-cultured at 25°C for 6 days. The experiment consisted of two 30-day leaching trials. A 42-day interval was set between the first and second 30-day leaching trials. This interval was set because after the first leaching trial, the microorganisms may have been disturbed and needed time to recover and regenerate. The 42-day interval provided sufficient time for the microorganisms to restabilize and adapt to environmental changes. This ensured relatively consistent conditions between the two leaching trials and improved the reliability and stability of the results. To simulate the water quality characteristics of surface runoff from nearby farmland, CO(NH2)2, KH2PO4, and C were added to the water-filled containers. 12 H 22 O 11 To prepare synthetic wastewater. During the experiment, the influent TN concentration was 4.47–13.85 mg / L, and NH4+ was... + -N concentration is 1.05–4.32 mg / L, NO3 - The -N concentration ranged from 0.34 to 11.26 mg / L. At the start of the experiment, 500 mL of synthetic runoff was pumped into each soil column. Synthetic runoff was pumped into the soil column every 6 days, for a total of 10 leaching tests. During the leaching experiments, the leaching solution was allowed to drain freely. After the synthetic wastewater was pumped into the soil column, all leaching solution samples were collected into 500 mL glass conical flasks within 72 hours, and the volume of the leaching solution was recorded in detail. Subsequently, the leaching solution was stored at 4°C, and water quality analysis was completed within 3 days. After all leaching tests were completed, soil samples from 0–20 cm depth were removed from each soil column and divided into two portions: one portion was used to determine the soil physicochemical properties, and the other portion was stored at -80°C for subsequent microbiological analysis.

[0058] 2. Soil and water quality analysis.

[0059] Soil SOC, TN, NH4 + -N, NO3 - -N, NO2- -N, AP, CEC, and pH were determined according to the standard method described by Lu Rukun (2000) (see: Lu Rukun. Soil Agricultural Chemical Analysis Methods [M]. Beijing: China Agricultural Science and Technology Press, 2000.). Soluble organic carbon (DOC) was extracted with deionized water, shaken for 30 min, centrifuged at 10000 rpm for 10 min, filtered through a 0.45 μm polyethersulfone membrane, and measured on a carbon and nitrogen analyzer (Vario EL III, Elementar, Langenselbold, Germany). At the end of the leaching experiment, biochar was separated from the soil using tweezers, washed, and characterized using Fourier transform infrared spectroscopy (FTIR) to compare the changes in surface functional groups of biochar before and after leaching. TN concentration in the influent and effluent was determined using the potassium persulfate oxidation method. NH4 in the influent and effluent... + -N、NO 3- -N and NO 2- -N concentrations were determined using a flow analyzer (AA3, Seal, Germany).

[0060] 3. Determination of nitrogen cycle functional genes.

[0061] DNA was extracted from soil samples according to the kit instructions. After extraction, the total DNA amount and purity were measured using a Qubit 4.0 instrument (ThermoFisher Scientific, Waltham, USA), ensuring a uniform DNA concentration of 20 ng / μl. -1 Real-time quantitative polymerase chain reaction (qPCR) was used to quantify N-cycle-related functional genes (nirS2, narG, nifH, nirS1, hzo, nirS3, gdhA, hao, nirK1, napA, amoB, nirK2, nosZ2, nasA, nirK3, amoA1, hzsB, nosZ1, amoA2). Primers for each gene are shown in Table 1. All qPCR reactions for each primer set were repeated three times, with each run including a non-template negative control. The initial isothermal phase was 95℃ for 5 min, followed by a 40-cycle phase, including denaturation at 95℃ for 15 s, annealing at 60℃ for 1 min, and extension at 72℃ for 20 s, during which fluorescence was read. The melting phase consisted of holding at 95℃ for 15 s, holding at 60℃ for 1 min, and finally holding at 95℃ for 15 s, with continuous sampling to monitor fluorescence changes.

[0062] Table 1 Primer sequences for nitrogen cycle-related functional genes

[0063]

[0064]

[0065] Wherein, W = A or T; S = C or G; R = A or G; Y = C or T; K = G or T; M = A or C; B = C, G, or T; D = A, G, or T; H = A, C, or T; V = A, C, or G; N = A, C, G, or T; I = hypoxanthine. Furthermore, in the sequence listing, except for sequences SEQ ID NO.19 and SEQ ID NO.20, N = A, C, G, or T; I in SEQ ID NO.19 and SEQ ID NO.20 is represented by N in the sequence listing, I = hypoxanthine, and I can pair with adenine (A), cytosine (C), and uracil (U).

[0066] After passing the initial DNA test, samples were added to 384-well plates as sample source plates, while primers and qPCR reagents were added to another 384-well plate as primer source plates. Using a high-throughput automated microdispenser, the SmartChip Multisample Nanodispenser (Takara Biomedical Technology), reagents from both sample and primer source plates were added to the microwells of a high-throughput qPCR chip, the SmartChip MyDesign Chip (Takara Biomedical Technology, Clontech). qPCR reactions and fluorescence signal detection were performed using the SmartChip Real-Time PCR System (WaferGen Biosystems USA), which automatically generated amplification and melting curves.

[0067] Absolute quantitative information of the 16S rRNA gene was obtained by real-time PCR (Roche, LightCycler 480 II). Based on the absolute quantification of the 16S rRNA gene, the absolute quantitative information of each gene in each sample was obtained using the following formula:

[0068] 16S rRNA relative quantification / 16S rRNA absolute quantification = gene relative quantification / gene absolute quantification.

[0069] 4. Data processing.

[0070] Data were analyzed using SPSS 22.0 software. One-way ANOVA and Tukey's HSD (p<0.05) were used to test for statistically significant differences in TN removal efficiency, soil properties, and nitrogen cycling functional genes among different treatments.

[0071] In the R language environment, Pearson correlation analysis was performed to explore the correlation between TN removal efficiency and nitrogen cycle functional genes.

[0072] A multivariate regression tree (MRT) was constructed using the mvpart package in R software to determine the most important soil properties affecting the copy numbers of nasA, nirK2, nirK3, and nosZ2.

[0073] 5. Results and Analysis.

[0074] 5.1 Soil and biophysical-chemical properties of carbon.

[0075] The physicochemical properties of the soil and biochar are shown in Table 2. It is evident that the physicochemical properties of different biochars vary. Specifically, GBC has higher TOC and CEC than FBC and SBC. FBC has higher TN and BET specific surface areas than GBC and SBC. SBC has higher NH4+. + -N、NO 3- The content of -N and AP is higher than that of FBC and GBC, but its pH value is significantly lower than that of FBC and GBC.

[0076] Table 2 Physicochemical properties of soil and biochar

[0077]

[0078] 5.2 Soil properties.

[0079] Soil properties obtained from different treatment groups are shown in Table 3. Compared with the control, biochar addition increased soil pH, AP, and CEC (Table 3, p<0.05), with GBC showing the highest increase and SBC showing the lowest. Biochar application also increased soil SOC and TN content, with the SBC treatment showing the lowest SOC and TN content. FBC and GBC addition significantly increased soil DOC, but SBC had no effect on DOC.

[0080] Table 3 Soil properties of different treatment groups

[0081]

[0082] Note: Different lowercase letters indicate significant differences between treatments (P<0.05). Control: control; FBC: fresh biochar; GBC: greenhouse aged biochar; SBC: soil aged biochar.

[0083] 5.3 Denitrification efficiency of different treatment groups.

[0084] Obtain TN and NO from the influent and effluent of different treatment groups 3- -N, NH4 + -N and NO 2--N concentration changes are as follows Figure 2 As shown, the TN removal efficiency of different treatment groups is compared as follows: Figure 3 As shown in the figure. It can be seen that TN and NO in the leachate... 3- -N and NH4 + The average concentrations of -N were 0.15 mg·L⁻¹. -1 ~26.4 mg·L -1 0.11 mg·L -1 ~24.2 mg·L -1 and 0.009 mg·L -1 ~0.18 mg·L -1 The control concentration was the highest, and the GBC concentration was the lowest. (See [reference]). Figure 2 (a) Figure 2 (b) and Figure 2 (c) In the first leaching cycle, NO 2- The average concentration of -N was 0.004 mg·L⁻¹. -1 ~0.09 mg·L -1 The SBC concentration was the lowest in the second leaching cycle, while NO2 was the highest in the third cycle. - The average concentration of -N was 0.0006 mg·L⁻¹. -1 ~0.01mg·L -1 Among them, FBC concentration was the lowest, see Figure 2 (d) The TN removal efficiency of the control treatment (-13.2% to 4.37%) was lower than that of the GBC (82.7% to 83.3%), FBC (59.0% to 61.4%) and SBC (27.0% to 29.8%) treatments.

[0085] 5.4 Changes in functional groups on the surface of biochar.

[0086] Fourier transform infrared spectra of different treatment groups before and after leaching experiments are shown below. Figure 4 As shown. Compared to FBC, GBC at 3400cm -1 and 1095cm -1 The surrounding area exhibits even higher peaks, with 3400 cm⁻¹ being the highest. -1 The nearby peak corresponds to the -OH stretching vibration, 1095 cm⁻¹ -1 The nearby peaks correspond to the -COOH, C=O, and COC stretching vibrations. Compared to the initial state at the time of the experiment, the functional groups on the FBC and GBC surfaces underwent significant changes after the leaching experiment; see [link to relevant documentation]. Figure 4 (a) and Figure 4 (b) in the text. However, the leaching test had no effect on the surface functional groups of SBCs; see [reference needed]. Figure 4 (c) of the text. All biochar was heated at 3200 cm⁻¹. -1 ~3500cm-1 A broad phenolic hydroxyl (-OH) peak appeared within the range. After the addition of FBC and GBC, the peak at 3400 cm⁻¹ was observed. -1 The peaks around the left and right become sharper, indicating an increase in amino (-NH2) content, with GBC showing a more pronounced change than FBC. After the addition of FBC, the peak at 1798 cm⁻¹... -1 The carbonyl (C=O) bond disappears at this point because the carboxylic acid compound (-COOH) reacts with -NH₂ to form an amide bond (-CONH). Similarly, in FBC and GBC, 1442 cm⁻¹ represents -COOH. -1 The peak also significantly decreased after reacting with the amino group. In GBC, it is located at 1095 cm⁻¹. -1 The broad wavelength range at 1587 cm⁻¹ is attributed to the stretching vibrations of -COOH, C=O, and ether groups (COC), and also decreased after leaching tests. Notably, GBC at 1587 cm⁻¹... -1 A new peak appeared at 1075 cm⁻¹, which is attributed to the NH bond in -CONH. Furthermore, FBC showed a peak at 1075 cm⁻¹. -1 A new peak was also observed, indicating the formation of CN bonds. This suggests that, compared to other biochars, greenhouse-aged biochar (GBC) has a richer abundance of oxygen-containing acidic functional groups, all of which react with nitrogen, promoting nitrogen adsorption.

[0087] 5.5 Analysis of nitrogen cycle functional genes and their underlying mechanisms affecting denitrification efficiency.

[0088] Obtain soil gene copy number pairs for different treatment groups, for example Figure 5 As shown, biochar application alters nitrogen cycle genes, but this effect depends on the gene and the type of biochar. Specifically, the copy numbers of the nirS2, narG, nifH, nirS1, hzo, nirS3, gdhA, and hao genes did not change significantly with biochar application. See [link to relevant documentation]. Figure 5 (a) In this context, compared to the control, the addition of FBC and GBC significantly increased the copy numbers of genes such as nirK1, napA, and amoB, while the addition of SBC significantly decreased the copy numbers of these genes. See [reference needed]. Figure 5 (b) Furthermore, GBC and FBC treatments increased the copy numbers of the nirK2, nosZ2, nasA, and nirK3 genes, while no such effect was observed in the SBC treatment. Compared to the control, the addition of GBC significantly increased the copy numbers of the amoA1, hzsB, and nosZ1 genes (p<0.05), while the copy number of the amoA2 gene reached the highest level in the FBC treatment.

[0089] Based on this, Pearson correlation analysis was performed to obtain the relationship between nitrogen cycle functional genes and TN removal efficiency, such as... Figure 6As shown, the copy numbers of NasA, nirK2, nirK3, and nosZ2 genes are significantly positively correlated with TN removal efficiency. Figure 6 Multiple regression tree analysis of the influence of soil properties on nitrogen cycle genes, as shown below. Figure 7 As shown, higher copy numbers of nasA, nirK2, nirK3, and nosZ2 in FBC and GBC are associated with higher DOC concentrations. Figure 7 The copy numbers of nasA, nirK2, and nosZ2 are higher in GBC than in FBC, which is related to the higher CEC in GBC.

[0090] In summary, GBC reduces TN and NO. 3- -N and NH4 + -N leaching is superior to other treatments; see [link to treatment]. Figure 2 (a) Figure 2 (b) and Figure 2 (c) In this experiment, the transparent greenhouse was subjected to intense sunlight and high temperatures during the summer, reaching a maximum temperature of 40–50°C. The intense sunlight and high temperatures oxidized the aromatic and aliphatic carbon on the surface of the biochar, leading to the formation of oxygen-containing acidic functional groups (such as carboxyl and hydroxyl groups). FTIR data support this hypothesis. Compared to FBC, GBC showed better performance at 3400 cm⁻¹. -1 and 1095cm -1 The surrounding area exhibits even higher peaks, with 3400 cm⁻¹ being the highest. -1 The nearby peak corresponds to the -OH stretching vibration, 1095 cm⁻¹ -1 The nearby peaks correspond to the -COOH, C=O, and COC stretching vibrations. The abundance of oxygen-containing acidic functional groups in GBC implies that it possesses more negatively charged sites (i.e., deprotonated oxygen-containing surface groups), thereby enhancing its resistance to NH4+. + -N adsorption capacity.

[0091] Results of gene assays targeting nitrogen cycle function showed that the copy numbers of three key genes, nasA, nirK2, and nosZ2, reached their maximum values ​​after GBC application. Importantly, the copy numbers of these three genes exhibited a significant positive correlation with TN removal efficiency. Figure 6 The nasA gene is involved in the assimilation of nitrate reduction, which is the process by which NO is reduced. 3- -N is converted to NO 2- -N. nirK2 and nosZ2 are key genes in the denitrification process, with nirK2 responsible for the second step of denitrification, namely NO. 2--N is converted to NO; nosZ2 is responsible for the final step of denitrification, which is the conversion of nitrous oxide (N2O) to nitrogen gas (N2). The increased number of nasA, nirK2, and nosZ2 genes in GBC indicates that GBC can significantly enhance the assimilation of nitrate reduction and denitrification rates, thereby accelerating NO production. 3- -N and NO 2- The conversion process of -N.

[0092] Furthermore, the above results also showed that, compared with other treatment groups, the higher nasA, nirK2, and nosZ2 gene counts in the GBC treatment group were associated with higher DOC and AP concentrations in the soil. This indicates that after greenhouse aging, residual DOC in biochar can still effectively stimulate the activity of microorganisms carrying nasA, nirK2, and nosZ2 genes. Notably, although the initial AP content of GBC was much lower than that of FBC and SBC (Table 2), the AP content in the soil after GBC application actually exceeded that of the soils treated with FBC and SBC (Table 3). The increased number of negatively charged surface functional groups (-COOH and -OH) in GBC reduced the adsorption of phosphate in the soil, thereby increasing the AP concentration. In addition, compared with FBC, the increased number of nasA, nirK2, and nosZ2 genes in GBC was also closely related to the improvement of CEC (Chemical Emission Capacity). Figure 7The -OH groups in carboxylic and phenolic groups in GBC can effectively adsorb and accommodate more exchangeable cations through the deprotonation process of organic functional groups. This directly explains why GBC exhibits a higher CEC (Table 1). Similarly, Lago et al. (2021) used a partial least squares regression (PLSR) model to predict the CEC of biochar and pointed out that phenolic and carboxylic acid groups are the main functional groups leading to the increase in biochar CEC (see: Lago, BC, Silva, CA, Melo, LCA, Morais, EGd, 2021. Predicting biochar cation exchange capacity using Fourier transform infrared spectroscopy combined with partial leastsquare regression. Sci Total Environ 794, 148762.). The oxyacid functional groups in GBC can act as both electron conductors and electron shuttles, meaning they can facilitate electron transfer to microorganisms encoding the nasA, nirK2, and nosZ2 genes. Furthermore, the increase in CEC also increases the number of exchangeable cations, which not only improves the availability of substrate nutrients but also provides richer nutrients for microorganisms encoding the nasA, nirK2, and nosZ2 genes in GBC. In summary, GBC application increases the copy number of the nasA, nirK2, and nosZ2 genes by increasing CEC, DOC, and AP levels in riparian soils. This increase in gene numbers further promotes nitrate reduction and denitrification processes, ultimately significantly improving TN removal efficiency.

[0093] in conclusion

[0094] (1) Both fresh biochar and different types of aged biochar can improve the TN removal efficiency of riparian zones, but the effect depends on the type of biochar. Compared with the control, the riparian zone with added GBC has the highest TN removal efficiency, while the riparian zone with added SBC has the lowest TN removal efficiency.

[0095] (2) The application of GBC increases the copy number of nasA, nirK2 and nosZ2 genes by increasing the content of CEC, DOC and AP in riparian soil, thereby ultimately improving the efficiency of TN removal in the riparian zone.

[0096] (3) Compared with freshly prepared or soil-aged biochar, greenhouse-aged biochar has greater potential for improving TN removal efficiency in riparian zones.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for improving nitrogen removal efficiency in riparian wetlands, comprising the following steps: 20% to 40% by volume of greenhouse-aged biochar was mixed into the soil in the riverbank zone to obtain a soil-biochar mixture; The topsoil of the riparian zone was replaced with a soil-biochar mixture, wherein the thickness of the topsoil of the riparian zone was ≤40cm. Improving nitrogen removal efficiency in riparian wetlands includes increasing the content of nitrogen cycling functional genes in riparian soils. The nitrogen cycle functional gene is NASA , nirK2 and / or nosZ2 ; The greenhouse-aged biochar is obtained through the following steps: Fresh biochar is placed in a greenhouse and exposed to natural sunlight for 2 to 4 consecutive years.

2. The method for improving nitrogen removal efficiency in riparian wetlands according to claim 1, characterized in that, After replacing the topsoil of the riverbank, compaction is carried out.

3. The method for improving nitrogen removal efficiency in riparian wetlands according to claim 1, characterized in that, The greenhouse-aged biochar has a particle size of < 2 mm.

4. The method for improving nitrogen removal efficiency in riparian wetlands according to claim 1, characterized in that, After replacing the topsoil of the riverbank with a soil-biochar mixture, quartz sand is laid on the surface of the soil-biochar mixture.

5. The method for improving nitrogen removal efficiency in riparian wetlands according to claim 4, characterized in that, The thickness of the paving is 4-6 cm.

6. The method for improving nitrogen removal efficiency in riparian wetlands according to any one of claims 1 to 5 reduces TN and NO3 in runoff water. - -N and / or NH4 + Applications of -N leaching.

7. The method for improving nitrogen removal efficiency in riparian wetlands according to any one of claims 1 to 5 is used to increase the content of nitrogen cycling functional genes in riparian soils.

8. The application according to claim 7, characterized in that, The nitrogen cycle functional gene is NASA , nirK2 and / or nosZ2 .

9. The method for improving nitrogen removal efficiency in riparian wetlands according to any one of claims 1 to 5, in enhancing the rate of assimilation of nitrate reduction and / or denitrification.

Images