Method for improving denitrification efficiency of riparian zone wetland and application

Abstract

The invention provides a method for improving the denitrification efficiency of a riparian wetland and application, and belongs to the technical field of environmental engineering. According to the method for improving the denitrification efficiency of the riparian zone wetland, the removal efficiency of the riparian zone wetland on total nitrogen (TN) in surface runoff water can be effectively improved, and water eutrophication caused by the fact that underground water is polluted by nitrogen in a leaching and runoff loss mode is prevented. Moreover, compared with other types of biochar (fresh biochar and soil aging biochar), the method for improving the denitrification efficiency of the riparian wetland, disclosed by the invention, has the advantages that the greenhouse aging biochar is specifically used as a material, so that a better treatment effect is obtained in the aspect of reducing leaching of TN, NO3 <->-N and / or NH4 < + >-N in a runoff water body; and in the aspect of biological factors, the copy number of nitrogen cycle functional genes in the riparian wetland soil is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental engineering, and in particular relates to a method for improving the denitrification efficiency of a riparian wetland and an application thereof. Background Art

[0002] Improper and excessive application of fertilizers in agriculture leads to rapid loss of nitrogen through ammonia volatilization, nitrogen-containing gases, leaching, and runoff. Among them, leaching and runoff account for nearly 30% of fertilizer application. A large amount of nitrogen lost through leaching and runoff pollutes groundwater and causes eutrophication of water bodies, posing a threat to human health. Therefore, there is an urgent need to develop effective strategies to reduce nitrogen leaching and runoff losses to water bodies.

[0003] Riparian zones are located at the interface between terrestrial and aquatic ecosystems and have been shown to prevent nitrogen loading from entering freshwater areas. Mayer et al. conducted a meta-analysis of 88 studies in 2007 and reported that the average nitrogen removal efficiency of riparian zones was 67.5% (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 a source of nitrogen pollution to water bodies (see: Inamdar, S., 2007. Exports of dissolved ammonium (NH 4 + )during storm events across multiple catchments in a glaciatedforested watershed.Environmental Monitoring and Assessment 133(1),347-363.). Therefore, new methods are needed to improve the efficiency of nitrogen removal in riparian zones. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application for improving the denitrification efficiency of riparian wetlands. The method for improving the denitrification efficiency of riparian wetlands described in the present invention can effectively improve the removal efficiency of TN in surface runoff water bodies by riparian wetlands, prevent nitrogen from polluting groundwater through leaching and runoff loss, and cause eutrophication of water bodies, posing a threat to human health.

[0005] The present invention provides a method for improving the denitrification efficiency of riparian wetlands, comprising the following steps:

[0006] Mixing 20% ​​to 40% by volume of greenhouse aged biochar into riparian soil to obtain a soil-biochar mixture;

[0007] The soil-biochar mixture was used to replace the topsoil in the riparian zone, the thickness of which was ≤ 40 cm.

[0008] Preferably, compaction is performed after replacing the topsoil in the riparian zone.

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

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

[0011] The fresh biochar was placed in a greenhouse and exposed to natural light for 2 to 4 consecutive years.

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

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

[0014] The present invention also provides a method for improving the denitrification efficiency of riparian wetlands according to the above technical solution to reduce TN and NO in runoff water. 3 - -N and / or NH 4 + -N leaching application.

[0015] 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 increasing the content of nitrogen cycle functional genes in riparian soil.

[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 assimilated nitrate reduction rate and / or the denitrification rate.

[0018] The present invention provides a method for improving the denitrification efficiency of riparian wetlands, which can effectively improve the removal efficiency of total nitrogen (TN) in surface runoff water bodies by riparian wetlands, and prevent nitrogen from polluting groundwater through leaching and runoff loss, leading to eutrophication of water bodies. Moreover, compared with other types of biochar (fresh biochar and soil aged biochar), the method for improving the denitrification efficiency of riparian wetlands of the present invention uses greenhouse aged biochar as a material, which not only reduces TN and NO in runoff water bodies, but also reduces the nitrogen content of TN and NO in runoff water bodies. 3- -N and / or NH 4 + -N leaching was better, and the copy number of nitrogen cycle functional genes in the riparian wetland soil was significantly improved in terms of biological factors. In particular, both the nasA gene involved in the assimilation of nitrate reduction and the key genes nirK2 and nosZ2 in the denitrification process reached the maximum copy number through the method for improving the denitrification efficiency of riparian wetlands of the present invention, proving that the method for improving the denitrification efficiency of riparian wetlands of the present invention using greenhouse aged biochar significantly enhances the assimilation of nitrate reduction and denitrification rates compared to other biochars, thereby further accelerating NO 3- -N and NO 2- -N conversion process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 A schematic diagram of the leaching test structure provided by the present invention;

[0021] Figure 2 The TN and NO in the inlet and outlet water of different treatment groups provided by the present invention 3- -N, NH 4 + -N and NO 2- -N concentration variation diagram;

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

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

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

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

[0026] Figure 7 This is a multiple regression tree analysis diagram of the impact of soil characteristics on nitrogen cycle genes provided by the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a method for improving the denitrification efficiency of riparian wetlands, comprising the following steps: mixing 20% ​​to 40% by volume of greenhouse aged biochar into riparian soil to obtain a soil-biochar mixture; using the soil-biochar mixture to replace the surface soil of the riparian zone, wherein the thickness of the surface soil of the riparian zone is ≤40 cm.

[0028] In a specific embodiment, the method for improving the denitrification efficiency of riparian wetlands can effectively improve the removal efficiency of total nitrogen in surface runoff water bodies in riparian wetlands, and prevent nitrogen from polluting groundwater through leaching and runoff loss, leading to eutrophication of water bodies. In a specific embodiment, after replacing the surface soil of the riparian zone, a compaction operation is performed. In a specific embodiment, the compaction degree of the compaction operation is to restore the original bulk density of the riverbank soil. In a specific embodiment, the particle size of the greenhouse aged biochar is <2 mm.

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

[0030] The fresh biochar was placed in a greenhouse and exposed to natural light 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, placed in a carbonization furnace, nitrogen is introduced for 1 hour, and then the temperature is raised from room temperature to 550°C at a rate of 15°C / min, and the temperature is kept constant 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 light intensity of the fully transparent glass greenhouse at noon on a sunny day in summer can be 800 to 1000 watts / square meter, and the light intensity at noon on a sunny day in winter can be 300 to 500 watts / square meter, and the annual average total solar radiation can be 1100 to 1400 kWh / square meter.

[0032] In a specific embodiment, after replacing the surface soil of the riparian zone with the soil-biochar mixture, quartz sand is laid on the surface of the soil-biochar mixture. In a specific embodiment, laying quartz sand can prevent the soil / soil-biochar mixture from being washed away. In a specific embodiment, the thickness of the laying is 4 to 6 cm.

[0033] The present invention also provides a method for improving the denitrification efficiency of riparian wetlands according to the above technical solution to reduce TN and NO in runoff water. 3- -N and / or NH 4 + -N leaching application.

[0034] 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 increasing the content of nitrogen cycle functional genes in riparian soil. In a specific embodiment, the nitrogen cycle functional genes include nasA, nirK2 and / or nosZ2. In a specific embodiment, the nasA is a gene that promotes the encoding of assimilation of nitrate reduction. In a specific embodiment, the 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 assimilated nitrate reduction rate and / or the denitrification rate.

[0036] In order to further illustrate the present invention, a method for improving denitrification efficiency of riparian wetlands and its application provided by the present invention are described in detail below in combination with the accompanying drawings and embodiments, but they should not be understood 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 was collected from the riparian zone of the Zhuanghang Comprehensive Experimental Station of Shanghai Academy of Agricultural Sciences (30°53′N, 121°23′E). The area has a subtropical monsoon climate, with an average annual precipitation of 1191.5 mm, evaporation of 1236.8 mm, and an average annual temperature of 16.1°C. The total annual sunshine hours are 1900.2 h, and the frost-free period is 224.4 d. The total organic carbon (SOC), total nitrogen (TN), and ammonium nitrogen (NH4+) in the surface layer of the riparian zone (0-20 cm) are 4 + -N), nitrate nitrogen (NO 3- -N), nitrite nitrogen (NO 2- -N), 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 the wood is collected, it is washed, cut, dried and placed in a carbonization furnace, nitrogen is introduced for 1 hour, and then the temperature is raised from room temperature to 550°C at a rate of 15°C / min, and the highest temperature is kept constant for 2 hours to obtain fresh biochar (FBC). Take some of the fresh biochar prepared above and divide it into two parts: one part is buried in the riparian soil and aged naturally in the soil environment to obtain soil-aged biochar (SBC), and the other part is placed in a greenhouse and aged naturally in the sun to obtain greenhouse-aged biochar (GBC). The specific preparation process is as follows:

[0043] Soil aged biochar: 30 cm of topsoil was removed from the original riparian zone, and a 10 cm thick mixture of riparian soil + fresh biochar was filled at 20-30 cm (river bank soil: fresh biochar was 1:1). After the mixture was filled, the original riparian soil was continued to cover the surface layer of 0-20 cm and planted with turf. After 36 months, the biochar added to the riparian zone was collected, and the visible biochar particles were separated from the soil sample with tweezers. The hand-picked biochar particles were then suspended in distilled water at a ratio of 1:10 (w / v), and the solution was gently shaken to remove any adhering soil particles. The biochar was then rinsed four times with distilled water. The washing step should not have any effect on the properties of the biochar. Finally, the washed biochar particles were placed in a convection oven and dried at 60 ° C to obtain soil aged 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 specific surface area, pH value and elemental composition of FBC, SBC and GBC were also determined. 4 + -N,NO 3 - -N,NO 2 - -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 straw biochar 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 for leaching test in specially designed polyvinyl chloride (PVC) columns, each of which was 55 cm high and 12 cm in inner diameter. Figure 1 shown.

[0050] The experiment consisted of 4 treatments:

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

[0052] (2) application of fresh biochar (FBC);

[0053] (3) application of greenhouse aged biochar (GBC);

[0054] (4) Application of soil aged biochar (SBC).

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

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

[0057] Before starting the leaching test, deionized water was added to all soil columns until the water level reached 60% of the field capacity. The soil columns were then pre-incubated at 25 °C for 6 days. The experiment was divided into two 30-day leaching tests in total. The interval between the first 30-day leaching test and the second 30-day leaching test was 42 days. The reason for setting this interval is that after the first leaching test, the microorganisms may have been disturbed and time is needed for them to recover and regenerate. The 42-day interval provides enough time for the microorganisms to restabilize and adapt to environmental changes. This ensures that the conditions between the two leaching tests are relatively consistent and improves the reliability and stability of the results. In order to simulate the water quality characteristics of surface runoff from nearby agricultural fields, CO(NH 2 ) 2 , KH 2 PO 4 and C 12 H 22 O 11 During the experiment, the influent TN concentration was 4.47-13.85 mg / L, NH 4 + -N concentration is 1.05~4.32mg / L, NO 3 --N concentration was 0.34-11.26 mg / L. At the beginning of the experiment, 500 mL of synthetic runoff water was pumped into each soil column. Synthetic runoff was pumped into the soil column every 6 days, and a total of 10 leaching tests were conducted in this experiment. During the leaching experiment, 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 bottles within 72 hours, and the volume of the leaching solution was recorded in detail. Subsequently, the leaching solution was stored in a 4°C refrigerator, and water quality analysis was completed within 3 days. After all the leaching tests were completed, 0-20 cm of soil was taken from each soil column and divided into two parts: one was used to determine the physical and chemical properties of the soil, and the other was placed in a -80°C refrigerator for subsequent microbial analysis.

[0058] 2. Soil and water quality analysis.

[0059] Soil SOC, TN, NH 4 + -N,NO 3 - -N,NO 2 - -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 minutes, centrifuged at 10,000 rpm for 10 minutes, filtered through a 0.45 μm polyethersulfone filter, and determined on a carbon and nitrogen element analyzer (Vario EL III, Elementar, Langenselbold, Germany). At the end of the leaching experiment, biochar was separated from the soil using tweezers, and after cleaning, it was characterized using Fourier transform infrared spectroscopy (FTIR) to compare the changes in the surface functional groups of biochar before and after leaching. The TN concentration in the inlet and outlet water was determined by potassium persulfate oxidation. NH 4 + -N, NO 3- -N and NO 2- -N concentration was determined using a flow analyzer (AA3, Seal, Germany).

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

[0061] The soil sample DNA was extracted according to the kit instructions. After extraction, the total amount and purity of DNA were tested using the Qubit4.0 (ThermoFisher Scientific, Waltham, USA) instrument to ensure that the DNA concentration was uniformly diluted to 20 ng·μl -1Real-time quantitative polymerase chain reaction (qPCR) was used to quantify the N cycle-related functional genes (nirS2, narG, nifH, nirS1, hzo, nirS3, gdhA, hao, nirK1, napA, amoB, nirK2, nosZ2, nasA, nirK3, amoA1, hzsB, nosZ1, amoA2). The primers corresponding to each gene are shown in Table 1. All qPCR reactions for each primer set were repeated three times, and a non-template negative control was included in each run. The initial constant temperature stage was 95°C for 5 min, followed by a cycling stage of 40 cycles, including denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and extension at 72°C for 20 s, during which the fluorescence was read. The melting stage consisted of: first holding at 95°C for 15 s, then maintaining at 60°C for 1 min, and finally holding at 95°C for another 15 s, while continuous sampling was performed to monitor the fluorescence changes.

[0062] Table 1 Primer sequences of 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. In addition, in the sequence list, except for the 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 list, I=hypoxanthine, and I can form a pair with adenine (A), cytosine (C) and uracil (U).

[0066] The qualified DNA samples were added to a 384-well plate as a sample plate (Sample Sourceplate), and the primers and reagents used for qPCR were added to another 384-well plate as a primer plate (Assay Sourceplate). The sample plate and primer plate reagents were added to the microwells of the high-throughput qPCR chip SmartChip MyDesign Chip (Takara Biomedical Technology, Clontech) using the high-throughput automatic micro-addition device SmartChip Multisample Nanodispenser (Takara Biomedical Technology), and the qPCR reaction and fluorescence signal detection were performed in the SmartChip Real-Time PCR System (WaferGen Biosystems USA), and the amplification curve and melting curve were automatically generated.

[0067] The absolute quantitative information of 16S rRNA gene was obtained by fluorescence quantitative PCR (Roche, LightCycler 480Ⅱ), and the absolute quantitative information of each gene in each sample was obtained according to the absolute quantitative conversion of 16S rRNA gene. The formula is as follows:

[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 statistically significant differences in TN removal efficiency, soil properties, and nitrogen cycle 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] Multiple regression trees (MRTs) were constructed using the mvpart package in R software to identify the most important soil characteristics affecting the copy numbers of nasA, nirK2, nirK3, and nosZ2.

[0073] 5 Results and analysis

[0074] 5.1 Physicochemical properties of soil and biomass charcoal.

[0075] The physical and chemical properties of soil and biochar are shown in Table 2. It can be seen that the physical and chemical properties of different biochars are different. Specifically, the TOC and CEC of GBC are higher than those of FBC and SBC. The TN and BET specific surface area of ​​FBC are higher than those of GBC and SBC. The NH 4 + -N, NO 3- -N and AP contents were higher than those of FBC and GBC, but its pH value was significantly lower than that of FBC and GBC.

[0076] Table 2 Physical and chemical properties of soil and biochar

[0077]

[0078] 5.2 Soil properties.

[0079] The soil properties obtained from different treatment groups are shown in Table 3. Compared with the control, the addition of biochar increased soil pH, AP and CEC (Table 3, p < 0.05), with GBC having the highest increase and SBC having the lowest increase. The application of biochar also increased soil SOC and TN content, with SBC treatment having the lowest SOC and TN content. The addition of FBC and GBC 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 among different treatments (P<0.05). Control: control; FBC: fresh biochar; GBC: greenhouse aged biochar; SBC: soil aged biochar.

[0083] 5.3 Nitrogen removal efficiency of different treatment groups.

[0084] Obtain TN and NO in the inlet and outlet water of different treatment groups 3- -N, NH 4 + -N and NO 2- -N concentration changes as Figure 2 As shown in Figure 2, the TN removal efficiency of different treatment groups is compared. Figure 3 As shown. It can be seen that TN and NO in the leaching solution 3- -N and NH 4 + The average concentration of -N was 0.15 mg·L -1 ~26.4mg·L -1 , 0.11mg·L -1 ~24.2mg·L -1 and 0.009mg·L-1 ~0.18mg·L -1 , where the control concentration was the highest and GBC concentration was the lowest, see 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 ~0.09mg·L -1 , where SBC concentration was the lowest, while in the second leaching cycle NO 2 - The average concentration of -N was 0.0006 mg·L -1 ~0.01mg·L -1 , among which FBC has the lowest concentration, 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 biochar surface functional groups.

[0086] The Fourier transform infrared spectra of different treatment groups before and after the leaching test were obtained as follows Figure 4 Compared with FBC, GBC has a -1 and 1095cm -1 There are higher peaks near 3400cm -1 The peak near 1095 cm corresponds to the -OH stretching vibration. -1 The peaks near correspond to -COOH, C=O and COC stretching vibrations. Compared with the beginning of the test, the functional groups on the surface of FBC and GBC changed significantly after the leaching test, see Figure 4 (a) and Figure 4 However, the leaching test has no effect on the surface functional groups of SBC, see Figure 4 (c) All biochars are at 3200 cm -1 ~3500cm -1 A broad phenolic hydroxyl (-OH) peak appears in the range. After adding FBC and GBC, 3400cm -1 The peaks on the left and right become sharper, which indicates that the amino group (-NH 2 ) content increased, and the change of GBC was more obvious than that of FBC. After adding FBC, 1798cm -1 The carbonyl (C=O) bond disappears due to the carboxylic acid compound (-COOH) and -NH 2The reaction forms an amide bond (-CONH). Similarly, the 1442 cm-1 residue in FBC and GBC represents -COOH. -1 The peak at 1095 cm-1 in GBC is also significantly weakened after reacting with amino groups. -1 The broad band at 1587 cm-1 is attributed to the stretching vibrations of -COOH, C=O and ether groups (COC), which also decreased after the leaching test. -1 A new peak appeared at 1075 cm-1, which was attributed to the NH bond in -CONH. -1 A new peak was also observed at , which represents the formation of CN bonds. It can be seen that compared with other biochars, greenhouse aged biochar (GBC) has more abundant oxygen-containing acidic functional groups, which react with nitrogen and promote nitrogen adsorption.

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

[0088] The comparison of soil gene copy numbers in different treatment groups was obtained. Figure 5 As shown. It can be seen that the application of biochar changed the nitrogen cycle genes, but this effect depended on the type of gene and biochar. Specifically, the copy numbers of nirS2, narG, nifH, nirS1, hzo, nirS3, gdhA and hao genes did not change significantly due to the application of biochar, see Figure 5 (a) in Figure 2. Compared with the control, the copy numbers of genes such as nirK1, napA, and amoB increased significantly after adding FBC and GBC, while the copy numbers of these genes decreased significantly after adding SBC, see Figure 5 (b). In addition, GBC and FBC treatments increased the copy numbers of nirK2, nosZ2, nasA, and nirK3 genes, while this effect was not observed in the SBC treatment. Compared with the control, the addition of GBC significantly increased the copy numbers of amoA1, hzsB, and nosZ1 genes (p<0.05), while in the FBC treatment, the copy number of amoA2 gene reached the highest level.

[0089] On this basis, the Pearson correlation analysis between nitrogen cycle functional genes and TN removal efficiency was obtained as follows Figure 6 As shown. It can be seen that the gene copy number of NasA, nirK2, nirK3 and nosZ2 is significantly positively correlated with the TN removal efficiency ( Figure 6 ). Multiple regression tree analysis of the effects of soil characteristics on nitrogen cycle genes Figure 7 Higher gene copy numbers of nasA, nirK2, nirK3, and nosZ2 in FBC and GBC were associated with higher DOC concentrations ( Figure 7). The copy numbers of nasA, nirK2, and nosZ2 were higher in GBC than in FBC, which was associated with the higher CEC in GBC.

[0090] In summary, GBC has a significant effect on reducing TN and NO 3- -N and NH 4 + -N is superior to other treatments in terms of leaching, see Figure 2 (a) Figure 2 (b) and Figure 2 (c) in the figure. The transparent greenhouse in this experiment was subjected to strong sunlight and high temperatures in the summer, with the highest temperature reaching 40-50°C. Strong sunlight and high temperatures oxidized the aromatic and aliphatic C on the surface of biochar, leading to the formation of oxygen-containing acidic functional groups (such as carboxyl and hydroxyl). FTIR data support this hypothesis. Compared with FBC, GBC has a higher carbon content at 3400cm -1 and 1095cm -1 There are higher peaks near 3400cm -1 The peak near 1095 cm corresponds to the -OH stretching vibration. -1 The peaks near the -COOH, C=O and COC stretching vibrations correspond to the abundant oxygen-containing acidic functional groups in GBC, which means that it has more negatively charged sites (i.e., deprotonated oxygen-containing surface groups), thereby enhancing the affinity of GBC to NH 4 + -N adsorption capacity.

[0091] The results of the determination of nitrogen cycle functional genes showed that after GBC application, the copy numbers of the three key genes nasA, nirK2 and nosZ2 reached the maximum value. More importantly, there was a significant positive correlation between the copy numbers of these three genes and the TN removal efficiency ( Figure 6 nasA gene is involved in the assimilation of nitrate reduction, i.e. NO 3- -N is converted to NO 2- -N. nirK2 and nosZ2 are key genes in the denitrification process, among which nirK2 is responsible for the second step of denitrification, that is, NO 2- -N is converted into NO; nosZ2 is responsible for the final step of denitrification, which is to convert nitrous oxide (N 2 O) into nitrogen (N 2 The increase in the number of nasA, nirK2, and nosZ2 genes in GBC indicated that GBC could significantly enhance the assimilative nitrate reduction and denitrification rates, thereby accelerating NO 3- -N and NO 2- -N conversion process.

[0092] Moreover, the above research results also showed that the higher nasA, nirK2 and nosZ2 genes in the GBC treatment group were associated with higher DOC and AP concentrations in the soil compared with other treatment groups. This indicates that after greenhouse aging, the residual DOC in the biochar can still effectively stimulate the activity of microorganisms carrying nasA, nirK2 and nosZ2 genes. It is worth noting that although the initial AP content of GBC was much lower than that of FBC and SBC (Table 2), after the application of GBC, the AP content in the soil exceeded that of the soil treated with FBC and SBC (Table 3). The increase in the number of negatively charged surface functional groups (-COOH and -OH) in GBC reduced the adsorption of phosphate by the soil, thereby increasing the AP concentration. In addition, compared with FBC, the increase in the number of nasA, nirK2 and nosZ2 genes in GBC is also closely related to the increase in CEC ( Figure 7 ). The -OH contained in the carboxylic acid 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 that lead to the increase in biochar CEC (see: Lago, BC, Silva, CA, Melo, LCA, Morais, EGd, 2021. Predicting biochar cation exchange capacityusing Fourier transforminfrared spectroscopy combined with partial leastsquare regression. Sci Total Environ 794, 148762.). The oxygen-containing acid functional groups in GBC can act as both electron conductors and electron shuttles, which means that they can facilitate electron transfer to microorganisms encoding nasA, nirK2, and nosZ2 genes. In addition, the increase in CEC also increased the number of exchangeable cations, which not only improved the availability of substrate nutrients, but also provided more abundant nutrition for microorganisms encoding nasA, nirK2, and nosZ2 genes in GBC. In summary, the application of GBC increased the copy number of nasA, nirK2, and nosZ2 genes by increasing CEC, DOC, and AP content in riparian soil. The increase in the number of these genes further promoted the assimilative nitrate reduction and denitrification processes, ultimately significantly improving the removal efficiency of TN.

[0093] in conclusion

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

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

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

[0097] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for improving the denitrification efficiency of riparian wetlands, comprising the following steps: Mixing 20% ​​to 40% by volume of greenhouse aged biochar into riparian soil to obtain a soil-biochar mixture; The soil-biochar mixture was used to replace the topsoil in the riparian zone, the thickness of which was ≤ 40 cm.

2. The method for improving the denitrification efficiency of riparian wetlands according to claim 1, characterized in that: After replacing the topsoil in the riparian zone, compaction operations are performed.

3. The method for improving denitrification efficiency of riparian wetlands according to claim 1, characterized in that: The particle size of the greenhouse aged biochar is <2 mm.

4. The method for improving denitrification efficiency of riparian wetlands according to claim 1, characterized in that: The greenhouse aged biochar is obtained by the following steps: The fresh biochar was placed in a greenhouse and exposed to natural light for 2 to 4 consecutive years.

5. The method for improving denitrification efficiency of riparian wetlands according to claim 1, characterized in that: After replacing the topsoil of the riparian zone with the soil-biochar mixture, quartz sand was laid on the surface of the soil-biochar mixture.

6. The method for improving denitrification efficiency of riparian wetlands according to claim 5, characterized in that: The paving thickness is 4 to 6 cm.

7. The method for improving the denitrification efficiency of riparian wetlands according to any one of claims 1 to 6 reduces TN, NO 3- -N and / or NH4 + -N leaching application.

8. Use of the method for improving denitrification efficiency of riparian wetlands according to any one of claims 1 to 6 in increasing the content of nitrogen cycle functional genes in riparian soil.

9. The use according to claim 8, characterized in that: The nitrogen cycle functional genes include nasA, nirK2 and / or nosZ2.

10. Use of the method for improving denitrification efficiency of riparian wetlands according to any one of claims 1 to 6 in enhancing assimilative nitrate reduction rate and / or denitrification rate.

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