A soil microbial regulation method for preventing and controlling nitrate leaching in fluvo-aquic soil

Abstract

The present invention discloses a soil microbial regulation method for preventing and controlling nitrate nitrogen leaching from fluvo-aquic soil, belonging to the technical field of soil remediation and improvement. The present invention applies biogas slurry simultaneously with seed sowing, with the seeds and biogas slurry spaced 8 to 12 cm apart. By regulating the subsoil microbial community and nitrogen conversion activity and activating the nitrate nitrogen conversion activity of indigenous microorganisms, the present invention reduces the nitrate nitrogen load in the soil, reduces nitrate nitrogen leaching, and is safe and harmless to the soil, effectively ensuring crop yields.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation and improvement, and in particular to a soil microbial control method for preventing and controlling nitrate nitrogen leaching from fluvo-aquic soil. Background Art

[0002] Soil nitrogen exists in various forms in the soil, and microorganisms are the primary driving force behind the transformation of nitrogen forms. Nitrogen leached from soil primarily occurs as nitrate nitrogen. Microorganisms can gradually convert nitrate nitrogen into N₂O and N₂ through denitrification and into organic nitrogen through assimilation. Both processes can reduce the nitrate nitrogen content in the soil. For farmland soils with high nitrate nitrogen content, there is significant potential to reduce the soil nitrate nitrogen load and thus the risk of nitrogen leaching through microbial conversion of nitrate nitrogen. Therefore, clarifying the microbial ecological mechanisms of nitrate nitrogen leaching from farmland in my country and understanding the key limiting factors are prerequisites for reducing nitrate nitrogen leaching from farmland through microbial regulation.

[0003] Denitrification and nitrate assimilation are two main microbial-driven processes that reduce nitrate nitrogen content. Denitrification is a process in which microorganisms convert nitrate nitrogen (NO 3- ) is finally converted into molecular nitrogen (N2). The overall reaction process can be expressed as: 2NO 3- +10e - +12H + →N₂ + 6H₂O. Nitrate assimilation refers to the process by which nitrate is reduced to ammonium and then further converted to organic nitrogen. Nitrate assimilation involves three specific pathways: nitrate absorption, nitrate reduction to nitrite, and nitrite reduction to ammonium. The latter two steps occur intracellularly, and the resulting ammonium can participate in other metabolic processes, such as amino acid synthesis.

[0004] A comprehensive study of various studies has found that there are a large number of denitrifying microorganisms and nitrate nitrogen assimilating microorganisms with diverse species in farmland soils, which have strong nitrate nitrogen conversion potential. However, the number of these microorganisms decreases significantly with increasing soil depth, especially in the soil below the plow layer. The denitrification and nitrate nitrogen assimilation activities are low, and the nitrate nitrogen conversion capacity is insufficient. As a result, excessive nitrate nitrogen cannot be converted and accumulates in the soil, causing the risk of nitrate nitrogen leaching.

[0005] As our understanding of the microbial mechanisms of soil nitrogen transformation continues to deepen, numerous attempts have been made to mitigate nitrate leaching by manipulating microbial communities and activity. For example, one study found that the addition of straw increased nitrate fixation by approximately two orders of magnitude, but the addition of organic fertilizer did not have a significant effect (Takashi et al., 2001). In another example, Qin Shuping et al. artificially created a potential difference (-5V) to increase the number and activity of nitrate-converting microorganisms, thereby enhancing soil microbial denitrification and dissimilatory reduction to ammonium (DNRA) capacity, effectively reducing soil nitrate nitrogen content (Qin et al., 2019). However, these methods are costly, lack mechanized implementation methods, and are currently immature for field application, making them difficult to promote. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a soil microbial regulation method for preventing and controlling the leaching of nitrate nitrogen in fluvo-aquic soil. By applying biogas slurry and sowing seeds simultaneously, the nitrate nitrogen load in the soil can be effectively reduced, thereby reducing the leaching of nitrate nitrogen.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a soil microbial control method for preventing and controlling nitrate nitrogen leaching from fluvo-aquic soil. Biogas slurry application and seed sowing are carried out simultaneously, and the distance between the seeds and the biogas slurry is 8 to 12 cm.

[0009] Preferably, the biogas slurry is obtained by anaerobic fermentation of human and animal feces and / or crop straw after removing solid matter.

[0010] Preferably, the biogas slurry is applied at a depth of 18 to 22 cm.

[0011] Preferably, the biogas slurry application rate is 400-600 kg / mu.

[0012] Preferably, irrigation is carried out after the biogas slurry is applied and sowing is completed.

[0013] Preferably, the method is used in a wheat-corn planting system in a tidal dryland area of ​​North China, and the biogas slurry is applied when summer corn is sown.

[0014] Preferably, the land is rotary tilled and prepared when wheat is sown, with a rotary tillage depth of 10 to 15 cm, and the land is not rotary tilled and prepared when corn is sown.

[0015] Preferably, fertilization is performed after wheat harvest and land preparation and before corn sowing.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] The present invention applies biogas slurry simultaneously with seed sowing, which can increase the organic carbon content in deep field soil, regulate the deep soil microbial community and nitrogen conversion activity, and activate the conversion activity of indigenous microorganisms to nitrate nitrogen, thereby effectively reducing the nitrate nitrogen load in the soil and reducing nitrate nitrogen leaching.

[0018] The raw material of the present invention is organic waste (biogas slurry), which is widely available and safe and harmless to the soil. It does not require other chemical synthesis products, and its application cost is low and its effect is good. At the same time, it can be mechanized when combined with a "seed and fertilizer integrated machine", which has low operating costs and can effectively guarantee crop yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Soil column experiment and results, A is the soil column experiment design, B is the nitrate nitrogen content and change rate of each soil layer in the control group (N fertilizer) and the experimental group (N fertilizer + biogas slurry);

[0020] Figure 2 : Total bacterial count (16S rRNA) and denitrification gene (nirS, nirK, nosZ) abundance in different soil layers;

[0021] Figure 3 : Relative abundance of nitrate-reducing and ammonifying functional microorganisms in different soil layers (bubble chart) and the change rate between biogas slurry treatment and control treatment (bar chart);

[0022] Figure 4 : Field application process and field nitrate leaching effect;

[0023] Figure 5 : The mechanism of action of biogas slurry in regulating microbial nitrogen transformation to reduce nitrate nitrogen leaching. DETAILED DESCRIPTION

[0024] The present invention provides a soil microbial control method for preventing and controlling nitrate nitrogen leaching from fluvo-aquic soil. Biogas slurry application and seed sowing are carried out simultaneously, and the distance between the seeds and the biogas slurry is 8 to 12 cm.

[0025] The soil microbial regulation method described in the present invention is applicable to fluvo-aquic soil regions. Fluvo-aquic soils are semi-hydrogenous soils that develop from alluvial deposits of rivers rich in or without carbonates, are affected by groundwater phreatic action, and are formed through tillage and maturation. Nitrogen leaching in fluvo-aquic soil regions is a serious problem, and this leaching primarily occurs during the summer corn crop. This method, developed in response to the problems and characteristics of fluvo-aquic soil regions, can enhance the nitrate-nitrogen conversion activity of soil microorganisms during the summer corn crop, reducing the nitrate-nitrogen load in the soil and ultimately reducing nitrate-nitrogen leaching.

[0026] The biogas slurry application and seed sowing are performed in staggered rows. Alternatively, a "seed-fertilizer integrated machine" can be used to mechanize simultaneous biogas slurry application and seed sowing. The spacing between the seeds and biogas slurry is preferably 9-11 cm, more preferably 10 cm. During biogas slurry application and seed sowing, the spacing should be controlled to prevent direct contact between the seeds and the biogas slurry.

[0027] The biogas slurry described herein is obtained by anaerobic fermentation of human and animal feces and / or crop straw, followed by removal of solid matter. The livestock and poultry feces herein include chicken, cow, pig, and sheep manure, and the crop straw herein includes wheat, rice, corn, potatoes, rapeseed, and cotton. As an optional embodiment, the biogas slurry described herein is obtained by anaerobic fermentation of human and animal feces and / or crop straw at 25-40°C. After removal of solid matter from the fermentation product, the resulting bright brown liquid is obtained.

[0028] The biogas slurry application depth of the present invention is 18 to 22 cm, preferably 19 to 21 cm, and more preferably 20 cm; the biogas slurry application rate is 400 to 600 kg / mu, preferably 450 to 550 kg / mu, and more preferably 500 kg / mu. Applying biogas slurry at this soil depth can effectively avoid activating urease activity in the surface soil and urease-producing microorganisms, thereby preventing nitrogen loss due to ammonia volatilization. Furthermore, this application depth allows for field operations using mechanical equipment.

[0029] In the present invention, irrigation is carried out after the biogas slurry is applied and the sowing is completed. The irrigation method and irrigation amount are determined according to local conditions.

[0030] The method of the present invention is preferably applicable to wheat-corn cropping systems in North China's aquic-dryland soils. In this system, both wheat and corn undergo straw shredding and return to the field; the soil is tilled to a depth of 10 to 15 cm, preferably 12 to 13 cm, during wheat sowing; fertilization is applied between wheat harvest and corn sowing, with the amount of fertilizer determined based on local conditions; no tillage is performed during corn sowing, and biogas slurry is applied during summer corn sowing to control nitrate leaching in wheat-corn cultivation sites in North China's aquic-dryland soils.

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] In the following examples, unless otherwise specified, all methods are conventional.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] In a specific embodiment of the present invention, the biogas slurry used was purchased from Jufeng Cattle Farm in Luquan District, Shijiazhuang. The basic properties of the biogas slurry are as follows: organic matter 3.05 g / kg, total nitrogen 0.083%, total phosphorus 0.002%, total potassium 0.098%, nitrate nitrogen 0.005%, and ammonium nitrogen 0.002%.

[0035] Example 1

[0036] This example uses a soil column experiment to study the effect of biogas slurry addition on the prevention and control of soil nitrate leaching and its mechanism of action:

[0037] Soil was collected from a long-term, site-specific experiment at the Agricultural Comprehensive Experimental Park of the Hebei Academy of Agriculture and Forestry Sciences. The soil is typically cultivated in a wheat-maize rotation system, using local management practices. Soil layers were collected from 0 to 60 cm (10 cm intervals) after wheat harvest. The soil was then refilled into a complete soil column (7.5 cm diameter and 60 cm height) in the laboratory according to the in situ soil bulk density for the experiment.

[0038] The experiment set up two treatments: control group and experimental group, each treatment was repeated 3 times (the experiment was as follows Figure 1 A). 1g of KNO3 solution was added to each of the two groups. The experimental group added biogas slurry at a depth of 20cm (500kg / mu, equivalent to 3.31g in the experiment). After the experimental setup was completed, the soil was incubated at 25°C for 60 days. 50ml of deionized water was added on days 10, 20, 35, and 50 to simulate precipitation. The filtrates were collected and their nitrate-nitrogen content was determined. After the incubation period, destructive sampling was performed on the soil column to determine the residual nitrate-nitrogen content in each layer.

[0039] The experimental results show that adding biogas slurry at 20cm can effectively reduce the nitrate nitrogen content in the 10-30cm soil layer ( Figure 1 B), and the total nitrate nitrogen leaching loss was reduced from 123mg to 85mg, a reduction of 31%. This shows that the addition of biogas slurry can effectively reduce nitrate nitrogen leaching loss.

[0040] The abundance of total microorganisms (expressed as 16S rRNA) and denitrifying microorganisms (expressed as nirS, nirK, and nosZ gene counts) was determined by real-time fluorescence quantitative PCR. Quantitative PCR primers are shown in Table 1.

[0041] Table 1 Information of primers used in quantitative PCR

[0042]

[0043] The quantitative PCR system is a 20 μl system, including Premix Ex Taq (TliRNaseH Plus, 2×, Takara Bio, Japan) (10 μl), forward primer (20 μM) (0.2 μl), reverse primer (20 μM) (0.2 μl), DNA template (20 ng / μl) (1 μl), and double-distilled water (8.6 μl). Amplification conditions were as follows: denaturation at 94°C for 5 min; 45 cycles of denaturation at 94°C for 30 s, annealing for 30 s (see Table 1 for annealing temperatures for each gene), extension at 72°C for 30 s, primer dimer melting at 80°C for 30 s, and fluorescence reading; a final extension at 72°C for 10 min. Quantitative PCR was performed using a CFX96 Optical Real-Time Detection System (Bio-Rad, Laboratories Inc., Hercules, CA, USA).

[0044] The results of quantitative PCR on the total amount of bacteria in each soil layer (expressed by 16S rRNA gene abundance) and the abundance of denitrification genes (nirS, nirK and nosZ) showed that adding biogas slurry could significantly increase the total amount of microorganisms ( Figure 2 ), indicating that the addition of biogas slurry increased the growth and reproduction rate of microorganisms. Microbial growth and reproduction will absorb and assimilate nitrogen, which means that the addition of biogas slurry may increase the assimilation of nitrate nitrogen by microorganisms, converting inorganic nitrogen into organic nitrogen, thereby reducing the content of nitrate nitrogen in the soil. At the same time, the addition of biogas slurry also significantly increased the abundance of denitrifying microorganisms ( Figure 2 ), which means that the denitrification capacity of the soil is effectively enhanced, and the denitrification of nitrate nitrogen is also beneficial to the reduction of the nitrate nitrogen content in the soil.

[0045] The bacterial community was determined by high-throughput sequencing technology. The specific primer pair 515F (SEQ ID NO: 9): 5'-GTGYCAGCMGCCGCGGTAA-3' and 806R (SEQ ID NO: 10): 5'-GGACTACNVGGGTWTCTAAT-3' was used to PCR amplify V3-V4 of the bacterial 16S rRNA. Then, high-throughput sequencing was performed using the Illumina HiSeq sequencing platform. The bacterial community composition and functional characteristics were analyzed using bioinformatics methods.

[0046] PCR amplification was performed in a 25 μl reaction system consisting of 12.5 μl of Premix Ex Taq (Takara Biotechnology, Japan), 0.5 μl of forward primer (20 μM), 0.5 μl of reverse primer (20 μM), 1 μl of DNA template (20 ng / μl), and 10.5 μl of double-distilled water. PCR reaction conditions were as follows: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, followed by extension at 72°C for 10 min.

[0047] Through the determination of soil microbial communities and functional potential, it was found that the application of biogas slurry significantly improved the nitrate reduction and ammonification processes of microorganisms in the 10-30 cm soil layer ( Figure 3 ), thereby reducing the nitrate nitrogen content in the soil.

[0048] The above results show that adding biogas slurry can effectively increase the total amount of soil microorganisms and the abundance of denitrifying microorganisms in the 10-30 cm soil, thereby improving the microbial absorption and conversion ability of nitrate nitrogen, reducing the content of nitrate nitrogen in the soil, and reducing the risk of nitrate nitrogen leaching.

[0049] Example 2

[0050] In this embodiment, a field verification experiment was carried out in the Agricultural Comprehensive Experimental Park of Hebei Academy of Agriculture and Forestry Sciences (the field treatment operations such as irrigation and fertilization of the experimental group and the control group were the same and were all carried out according to conventional methods):

[0051] Experimental group: The land was tilled to a depth of 12 cm when wheat was sown; fertilizer was applied between wheat harvest and corn sowing; the land was not tilled when corn was sown, and biogas slurry was applied at the same time as summer corn sowing. A "seed and fertilizer integrated seeder" was used to apply biogas slurry in a 20 cm soil layer, with a distance of 10 cm between seeds and biogas slurry, and the application rate of biogas slurry was 500 kg / mu.

[0052] Control group: The land was tilled to a depth of 12 cm when wheat was sown; fertilizer was applied between wheat harvest and corn sowing; the land was not tilled when corn was sown, and summer corn was sown.

[0053] After corn harvest, the nitrate nitrogen content in the 0-1m soil layer (one layer every 20cm) was tested. The results showed that the nitrate nitrogen content in each soil layer of the biogas slurry treatment was significantly lower than that of the control treatment ( Figure 4 ), indicating that this method can effectively prevent and control nitrate leaching in the field.

[0054] The corn yield was statistically analyzed and the results are shown in Table 2:

[0055] Table 2 Crop yields of control and biogas slurry treatment

[0056]

[0057] As shown in Table 2, there is no significant difference in crop yield compared with the control, and the yield per mu, average number of grains per ear and thousand-grain weight are slightly increased, indicating that the present invention can effectively ensure crop yield while reducing the problem of nitrate nitrogen leaching in the environment.

[0058] Through indoor experiments and field verification, it was found that applying biogas slurry 20 cm below the soil can reduce the soil nitrate nitrogen load by improving the ability of microorganisms to convert nitrate nitrogen, thereby achieving the purpose of reducing soil nitrate nitrogen leaching. At the same time, this method can also effectively maintain and increase crop yields and realize mechanized operations in the field.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A soil microbial control method for preventing and controlling nitrate nitrogen leaching from fluvo-aquic soil, characterized in that: The biogas slurry is applied and the seeds are sown simultaneously, and the distance between the seeds and the biogas slurry is 8 to 12 cm; The biogas slurry is applied at a depth of 18 to 22 cm; The biogas slurry application rate is 400-600 kg / mu; The method is used in a wheat-corn planting system in a North China tidal dryland. The biogas slurry is applied when the summer corn is sown. The land is rotary tilled to a depth of 10 to 15 cm when the wheat is sown, and no rotary tillage is performed when the corn is sown. Fertilization is performed between the time the wheat is harvested and the time the corn is sown. The basic properties of the biogas slurry are as follows: organic matter 3.05 g / kg, total nitrogen 0.083%, total phosphorus 0.002%, total potassium 0.098%, nitrate nitrogen 0.005%, and ammonium nitrogen 0.002%.

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