A porous composite microbial inoculant for promoting nitrate reduction to produce ammonium, and a preparation method and application thereof

Abstract

The present application relates to the technical field of soil improvement, and discloses a porous composite microbial agent for promoting nitrate reduction to produce ammonium, a preparation method and application thereof.The porous composite microbial agent is a modified porous mineral loaded with microorganisms having nitrate reduction capacity; the microorganisms having nitrate reduction capacity include at least one of Klebsiella pneumoniae, pseudomonas nitritireducen and Shewanella putrefaciens strain CN32.The porous composite microbial agent provided by the present application can promote the efficiency and rate of conversion of nitrate or nitrite to ammonium nitrogen, reduce nitrogen loss and greenhouse gas generation, has a compact structure, good stability and can continuously play a role; the porous composite microbial agent is applied to the process of strengthening soil nitrate dissimilatory reduction to ammonium, and can reduce soil nutrient loss and air pollution.

Description

Technical Field

[0001] This invention relates to the field of soil improvement technology, and in particular to a porous composite bacterial agent that promotes nitrate reduction to ammonium production, its preparation method, and its application. Background Technology

[0002] The application of nitrogen fertilizers, with nitrate nitrogen and ammonium nitrogen as the core components, plays an important role in ensuring food production. Compared with ammonium nitrogen, nitrate nitrogen is more likely to migrate with water bodies and produce gaseous nitrogen products, which can easily cause nutrient loss in farmland soil and environmental problems.

[0003] Nitrate nitrogen transformation in agricultural soils mainly includes two processes: denitrification and dissimilatory nitrate reduction (DNRA). Denitrification produces nitrogen gas and the greenhouse gas nitrous oxide, leading to nitrogen loss and air pollution. DNRA, on the other hand, converts nitrate into ammonium nitrogen, ensuring nitrogen availability in the soil. However, denitrification dominates in soil processes. Therefore, enhancing the role of DNRA in soil and promoting the conversion of nitrate to ammonium nitrogen is crucial for improving soil fertility efficiency, reducing nitrogen pollution, and developing high-quality agriculture.

[0004] CN 115974288 A discloses a method for enhancing the dissimilatory reduction of nitrate to ammonium by coupling an electric field with a magnetic field. The coupling effect of the electric and magnetic fields promotes the rapid start-up of the DNRA reactor and improves the stability of the system. At the same time, it increases the activity of DNRA bacteria and the abundance of the functional gene nrfA. However, this method is only applicable to aquatic environments and is difficult to promote and play a role in farmland soil regulation. In addition, it requires continuous consumption of electricity, resulting in high costs. CN 117070415 A discloses a highly efficient denitrifying strain with dissimilatory reduction to ammonium function and its application. This strain can achieve rapid removal of nitrate nitrogen in water while effectively reducing the production of nitrogen oxides. However, the application of this strain is still limited to aquatic environments and does not involve the improvement of soil nitrogen transformation.

[0005] It is evident that current research on enhancing the DNRA process mainly focuses on water treatment systems, and the methods primarily involve electrochemical enhancement and screening of new microbial strains. Further research is needed on methods for enhancing the DNRA process in soil. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a porous composite microbial agent.

[0007] The second objective of this invention is to provide a method for preparing this porous composite bacterial agent.

[0008] The third objective of this invention is to provide the application of this porous composite bacterial agent.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A first aspect of the present invention provides a porous composite microbial agent, wherein the porous composite microbial agent is a modified porous mineral loaded with microorganisms capable of reducing nitrates;

[0011] The microorganisms with nitrate-reducing ability include at least one of Klebsiella pneumoniae, pseudomonas nitritireducen, and Shewanella putrefaciens strain CN32.

[0012] In some embodiments of the present invention, Klebsiella pneumoniae (KP) and pseudodomonas nitritireducen were purchased from the China Marine Microbial Culture Collection Center, with accession numbers 1A18916 and 1A12677, respectively; Shewanella putrefaciens strain CN32 (CN32) was purchased from the Guangdong Provincial Microbial Culture Collection Center, with accession number 1.459.

[0013] In some embodiments of the present invention, the modified porous mineral is prepared from raw materials including: natural porous minerals, crop straw, active organic matter, composite fibers and water.

[0014] In some embodiments of the present invention, the particle size of the natural porous mineral is 0.5-2 mm.

[0015] In some specific embodiments of the present invention, the particle size of the natural porous mineral is 0.5-1.5 mm.

[0016] In some embodiments of the present invention, the specific surface area of ​​the natural porous mineral is 2-5 m². 2 / g.

[0017] In some specific embodiments of the present invention, the specific surface area of ​​the natural porous mineral is 2-3 m². 2 / g.

[0018] In some embodiments of the present invention, the particle size of the crop straw is less than or equal to 2 mm.

[0019] In some specific embodiments of the present invention, the particle size of the crop straw is 0.15-2 mm.

[0020] In some embodiments of the present invention, the specific surface area of ​​the modified porous mineral is 10-20 m². 2 / g.

[0021] In some specific embodiments of the present invention, the specific surface area of ​​the modified porous mineral is 15-20 m². 2 / g.

[0022] In some embodiments of the present invention, the natural porous mineral is selected from at least one of perlite, vermiculite, diatomite, volcanic rock, maifanite, and zeolite.

[0023] In some embodiments of the present invention, the active organic matter is selected from at least one of xylitol, maltitol, and sorbitol.

[0024] In this invention, substances rich in hydroxyl groups, such as xylitol, maltitol, and sorbitol, are selected as active organic matter. The hydroxyl functional groups therein can form intermolecular hydrogen bonds with active groups such as hydroxyl and carboxyl groups on composite fibers, the surface of microbial cells with nitrate-reducing ability, and the surface of modified porous minerals. This allows the raw materials for the preparation of porous composite bacterial agents to be firmly combined, thereby obtaining a porous composite bacterial agent with a compact structure and good stability.

[0025] In some embodiments of the present invention, the composite fiber is selected from at least one of lignin-based carbon fiber, polylactic acid-based composite fiber, and phenolic resin-based carbon fiber.

[0026] In some embodiments of the present invention, the crop straw is cereal crop straw.

[0027] In some specific embodiments of the present invention, the crop straw is selected from at least one of rice straw, corn straw, and wheat straw.

[0028] In this invention, cereal crop straw rich in cellulose, hemicellulose and lignin is selected as the raw material for the preparation of modified porous minerals. In the subsequent pyrolysis and carbonization process, it is more conducive to the formation of a biochar layer with good quality, good adsorption and good stability.

[0029] In some embodiments of the present invention, the modified porous mineral is prepared by a method comprising the following steps:

[0030] Natural porous minerals and crop straw are mixed and then pyrolyzed and carbonized to obtain porous minerals with a biochar layer.

[0031] The active organic matter, composite fiber and water are mixed, and the porous mineral with the biochar layer is added and dried to obtain the modified porous mineral.

[0032] In some embodiments of the present invention, after adding the porous mineral with the biochar layer, the mixture is further stirred for 20-40 minutes.

[0033] In some embodiments of the present invention, the mass ratio of the natural porous mineral to crop straw is (5-25):1.

[0034] In some specific embodiments of the present invention, the mass ratio of the natural porous mineral to crop straw is (7-20):1.

[0035] In some embodiments of the present invention, the heating procedure for pyrolysis carbonization is as follows: heating at 120-170°C for 30-60 minutes, and then heating to 700-850°C for 30-60 seconds.

[0036] In some specific embodiments of the present invention, the heating procedure for pyrolysis carbonization is as follows: heating at 130-160°C for 30-40 minutes, and then heating to 700-800°C for 30-50 seconds.

[0037] In some embodiments of the present invention, the mass ratio of the active organic matter, composite fiber, water and porous mineral with biochar layer is 1:(5-15):(60-110):(500-850).

[0038] In some specific embodiments of the present invention, the mass ratio of the active organic matter, composite fiber, water and porous mineral with biochar layer is 1:(8-13):(60-100):(500-800).

[0039] In some embodiments of the present invention, the drying temperature is 30-60°C and the time is 10-15 hours.

[0040] In some specific embodiments of the present invention, the drying temperature is 30-50°C and the time is 10-13 hours.

[0041] The second aspect of the present invention provides a method for preparing the porous composite bacterial agent described in the first aspect of the present invention, comprising the following steps:

[0042] The modified porous minerals were co-cultured with microorganisms capable of reducing nitrates to obtain the porous composite bacterial agent.

[0043] In some embodiments of the present invention, the density of the microorganisms with nitrate-reducing ability is OD. 600 =1-1.5; the solid-liquid ratio of the porous mineral to the microorganism with nitrate-reducing ability is 1g:(0.2-1)mL.

[0044] In some specific embodiments of the present invention, the density of the microorganisms with nitrate-reducing ability is OD. 600 =1-1.3; the solid-liquid ratio of the porous mineral to the microorganism with nitrate-reducing ability is 1g:(0.2-0.6)mL.

[0045] In some embodiments of the present invention, the co-culture further includes the use of a buffer solution with pH = 7.0; the concentration of the buffer solution is 40-60 mmol / L; and the volume ratio of the buffer solution to the microbial culture is (20-30):1.

[0046] In some specific embodiments of the present invention, the buffer solution comprises a borate-borax buffer solution.

[0047] In some embodiments of the present invention, the co-culture further includes the use of nutrients; the solid-liquid ratio of the nutrients to the microbial culture is 1g:(20-30)mL.

[0048] In some specific embodiments of the present invention, the nutritional components include sodium nitrate and sodium lactate in a mass ratio of 1:(1-2).

[0049] In some embodiments of the present invention, the co-culture temperature is 25-30°C and the time is 15-20 hours.

[0050] In some specific embodiments of the present invention, the co-culture temperature is 27-30°C and the time is 15-18 hours.

[0051] In some embodiments of the present invention, after the co-culture is completed, the process further includes centrifugation, solid-liquid separation to collect the solid phase, and obtaining a porous composite bacterial agent.

[0052] The third aspect of the present invention provides the application of the porous composite bacterial agent described in the first aspect of the present invention in enhancing the dissimilatory reduction of soil nitrate to ammonium.

[0053] In some embodiments of the present invention, the mass ratio of the porous composite microbial agent to the soil is 1:(10-20).

[0054] In some specific embodiments of the present invention, the mass ratio of the porous composite microbial agent to the soil is 1:(10-15).

[0055] In some embodiments of the present invention, the physicochemical properties of the soil include at least one of the following:

[0056] pH value is 4.0-8.0;

[0057] The total iron content is 15-30 g / kg;

[0058] The organic nitrogen content is 20-35 g / kg;

[0059] The cation exchange capacity is 5-15 cmol / kg.

[0060] In some specific embodiments of the present invention, the physicochemical properties of the soil include at least one of the following:

[0061] pH value is 5.0-7.0;

[0062] The total iron content is 15-20 g / kg;

[0063] The organic nitrogen content is 25-30 g / kg;

[0064] The cation exchange capacity is 5-10 cmol / kg.

[0065] The basic principles of this invention are explained as follows:

[0066] 1) This invention first uses natural porous minerals and crop straw as raw materials, and obtains porous minerals with biochar layers through pyrolysis and carbonization. These porous minerals with biochar layers have rich pore structures and large specific surface areas. On the one hand, the porous structure provides a large number of attachment sites for microorganisms with nitrate-reducing capabilities, enabling the microorganisms to exist stably in the inoculant and not easily lost. On the other hand, the adsorption effect of biochar can enrich nitrates in the soil, increasing their concentration around the microorganisms and increasing the contact opportunities between the microorganisms and the substrate, thereby promoting nitrate conversion. At the same time, this mineral-biochar composite structure can also improve soil aeration and water retention, increase soil porosity and respiration capacity, and create a suitable living environment for microorganisms.

[0067] 2) After mixing porous minerals with biochar layers with active organic matter, composite fibers and water, ① the composite fibers have high strength and stability, and can be connected with raw materials such as porous minerals with biochar layers and active organic matter through chemical bonds (such as covalent bonds, hydrogen bonds and π-π stacking interactions), which enhances the structural stability of the entire composite microbial agent and enables it to remain stable in the soil environment for a long time; in addition, carbon fibers have good electrical conductivity, which can promote electron transfer in the metabolic process of microorganisms. Electron transfer plays a crucial role in the reduction of nitrate to ammonium nitrogen. Composite fibers such as lignin-based carbon fibers can accelerate electron transfer, improve the efficiency of the nitrate reduction reaction, and thus promote the DNRA process, increasing the production of ammonium nitrogen. Secondly, active organic matter such as xylitol is a high-quality carbon source for the growth and metabolism of microorganisms with nitrate-reducing capabilities. In the soil environment, microorganisms utilize active organic matter for growth and reproduction, providing energy and material basis for the nitrate reduction process. Simultaneously, the presence of active organic matter can regulate the metabolic pathways of microorganisms. Studies have shown that suitable carbon sources can influence the metabolic direction of microorganisms, making nitrate-reducing bacteria more inclined to reduce nitrate to ammonium nitrogen via the DNRA pathway rather than undergoing denitrification to produce nitrogen gas and nitrous oxide, thereby reducing nitrogen loss during denitrification.

[0068] 3) By co-culturing microorganisms with nitrate-reducing ability with modified porous minerals, a porous composite microbial agent is obtained. The microorganisms with nitrate-reducing ability are loaded onto the modified porous minerals, thus possessing the ability to reduce nitrates to ammonium nitrogen. In the soil environment, the microorganisms can use carbon sources such as active organic matter for growth and metabolism, and in the process of metabolism, through a series of enzymatic reactions, reduce nitrates in the soil to ammonium nitrogen.

[0069] 4) The porous composite microbial agent also contains abundant organic matter, such as biochar, which directly replenishes the soil with organic carbon source and increases the content of soil organic matter. The active organic matter is partially utilized by microorganisms in the soil, and the part that is not completely metabolized will remain in the soil and become part of the soil organic matter. Therefore, the porous composite microbial agent can also play a role in increasing the soil organic carbon content and improving soil properties.

[0070] Compared with the prior art, the beneficial effects of the present invention are:

[0071] 1) The porous composite microbial agent provided by the present invention can promote the efficiency and rate of conversion of nitrate or nitrite to ammonium nitrogen, reduce nitrogen loss and greenhouse gas generation. The porous composite microbial agent has a compact structure, good stability and can play a continuous role.

[0072] 2) The method for preparing porous composite bacterial agent provided by the present invention has simple steps, low raw material cost, no need to use complicated instruments, and does not generate waste, making it environmentally friendly and suitable for industrial use.

[0073] 3) The porous composite microbial agent provided by this invention can be applied to enhance the dissimilatory reduction of soil nitrate to ammonium, reduce soil nutrient loss and air pollution, and when applied to the soil, it can also increase soil porosity and respiration capacity, increase soil organic carbon content, and improve soil properties. Attached Figure Description

[0074] Figure 1 This is a schematic diagram showing the change in nitrate content in Experiment Example 1;

[0075] Figure 2 This is a schematic diagram showing the change in nitrite content in Experiment Example 1;

[0076] Figure 3 This is a schematic diagram showing the change in ammonium nitrogen content in Experiment Example 1;

[0077] Figure 4 This is a schematic diagram showing the change in nitrite content in Experiment Example 2;

[0078] Figure 5 This is a schematic diagram showing the change in nitrous oxide content in Experiment Example 2;

[0079] Figure 6 This is a schematic diagram showing the change in ammonium nitrogen content in Experiment Example 2;

[0080] Figure 7 This is a schematic diagram showing the change in nitrate content in Experiment Example 3;

[0081] Figure 8 This is a schematic diagram showing the change in ammonium nitrogen content in Experiment Example 3;

[0082] Figure 9 This is a schematic diagram showing the change in dissolved organic carbon content in Experiment Example 3. Detailed Implementation

[0083] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and test examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0084] Example 1

[0085] This embodiment prepares a porous composite bacterial agent, and the steps are as follows:

[0086] S11, with a particle size of 0.5-1mm and a specific surface area of ​​2-4m². 21 kg of perlite ( / g) is placed in a cool, ventilated place for 1-2 days to ensure that the surface moisture is completely removed. After the rice straw is dried, it is processed into powder with a particle size of 0.15-2mm using a pulverizer.

[0087] S12. After uniformly mixing the treated perlite and rice straw powder, put them into a muffle furnace (wherein, the mass ratio of perlite to rice straw powder is 13:1), start the heating program, first heat at 150℃ for 35 minutes, then raise the temperature to 800℃ and continue heating for 40 seconds to obtain a porous mineral with a biochar layer.

[0088] S21. Accurately weigh 0.5g xylitol and 5g lignin-based carbon fiber into a 1.5L aluminum box, add 50mL deionized water, mix well, then add 400g of porous mineral with a biochar layer, seal the aluminum box, stir for 30min, and then place the sample in an oven at 40℃ for 12h to obtain modified porous mineral.

[0089] S31. Add 500 mL of anaerobic, sterile 50 mmol / L borate-borax buffer solution (pH = 7.0), 50 g of modified porous mineral, and 20 mL of OD to a large vial. 600 A KP bacterial culture with a concentration of 1.25 μg, 0.3 g sodium nitrate, and 0.5 g sodium lactate were mixed and placed in an incubator at 28°C for 18 hours. After incubation, the sample was centrifuged, and the precipitate was collected, yielding a sample with a specific surface area of ​​16.3 m². 2 / g porous composite bacterial agent.

[0090] Example 2

[0091] This embodiment prepares a porous composite bacterial agent, and the steps are as follows:

[0092] S11, with a particle size of 0.5-2mm and a specific surface area of ​​2-3m². 2 1 kg of perlite ( / g) is dried at room temperature for 1-2 days to ensure that the surface moisture is completely removed. After the rice straw is dried, it is processed into powder with a particle size of 0.15-2mm using a pulverizer.

[0093] S12. After uniformly mixing the treated perlite and rice straw powder, put them into a muffle furnace (wherein, the mass ratio of perlite to rice straw powder is 19:1), start the heating program, first heat at 160℃ for 35 minutes, then raise the temperature to 800℃ and continue heating for 45 seconds to obtain a porous mineral with a biochar layer.

[0094] S21. Accurately weigh 0.8g xylitol and 6.5g lignin-based carbon fiber into a 1.5L aluminum box, add 50mL deionized water, mix well, then add 400g porous mineral with biochar layer, seal the aluminum box, stir for 30min, and then place the sample in an oven at 40℃ for 12h to obtain modified porous mineral.

[0095] S31. Add 500 mL of anaerobic, sterile 50 mmol / L borate-borax buffer solution (pH = 7.0), 50 g of modified porous mineral, and 20 mL of OD to a large vial. 600 A KP bacterial culture with a concentration of 1.25 μg, 0.3 g sodium nitrate, and 0.5 g sodium lactate were mixed and placed in an incubator at 28°C for 18 hours. After incubation, the sample was centrifuged, and the precipitate was collected, yielding a specific surface area of ​​15.4 m². 2 / g porous composite bacterial agent.

[0096] Example 3

[0097] This embodiment prepares a porous composite bacterial agent, and the steps are as follows:

[0098] S11, with a particle size of 0.5-1.5mm and a specific surface area of ​​2-3m². 2 1 kg of perlite ( / g) is dried at room temperature for 1-2 days to ensure that the surface moisture is completely removed. After the rice straw is dried, it is processed into powder with a particle size of 0.15-2mm using a pulverizer.

[0099] S12. After uniformly mixing the treated perlite and rice straw powder, put them into a muffle furnace (wherein, the mass ratio of perlite to rice straw powder is 7:1), start the heating program, first heat at 130℃ for 35 minutes, then raise the temperature to 700℃ and continue heating for 45 seconds to obtain a porous mineral with a biochar layer.

[0100] S21. Accurately weigh 0.8g xylitol and 10g lignin-based carbon fiber into a 1.5L aluminum box, add 60mL deionized water, mix well, then add 450g porous mineral with a biochar layer, seal the aluminum box, stir for 30min, and then place the sample in an oven at 40℃ for 12h to obtain modified porous mineral.

[0101] S31. Add 500 mL of anaerobic, sterile 50 mmol / L borate-borax buffer solution (pH = 7.0), 50 g of modified porous mineral, and 20 mL of OD to a large vial. 600 A KP bacterial culture with a concentration of 1.25 μg, 0.3 g sodium nitrate, and 0.5 g sodium lactate were mixed and placed in an incubator at 28°C for 18 hours. After incubation, the sample was centrifuged, and the precipitate was collected, yielding a sample with a specific surface area of ​​15.8 m². 2 / g porous composite bacterial agent.

[0102] Experimental Example 1: Nitrate Conversion Experiment

[0103] Test method:

[0104] Add 50-80 mL of soil simulation solution to 100-150 mL vials to construct a microcosm culture system. The soil simulation solution includes: anaerobic borax-boric acid buffer, nitrate, and sodium lactate. The concentration of the borax-boric acid buffer is 50 mmol / L, pH = 6.8-7.0, the concentration of nitrate is 4.5-6.5 mmol / L, and the concentration of sodium lactate is 1.0-1.5 mmol / L. The experiment is divided into two groups: one is the experimental group, which adds 1-2 g of the synthesized porous composite bacterial agent; the other is the control group, which adds only KP bacterial solution. After adding the porous composite bacterial agent and bacterial solution, the vials are sealed and shaken well. Then, three vials from each group are placed in a constant temperature incubator and statically cultured at 28-30℃. The concentration changes of nitrate, nitrite, and ammonium nitrogen in the reaction system are measured every 12 hours.

[0105] The method for determining the concentrations of nitrate and nitrite is as follows: Mix the sample in the vial thoroughly, use a clean 5mL syringe to draw 1mL of the suspension into a centrifuge tube containing 4mL of deionized water, and extract on a shaker for 5min with the following extraction parameters: protected from light, 30℃, and 200rpm; after extraction, centrifuge the tube for 3min with the following centrifugation parameters: 25℃ and 6000rpm; after centrifugation, take the supernatant and filter it through a 0.22μm filter. Then, use an ion chromatograph to determine the nitrate and nitrite concentrations in the filtrate, and calculate the actual content of both in the extracted sample based on the nitrate and nitrite standard lines.

[0106] The method for determining the concentration of ammonium nitrogen is as follows: Mix the sample in the vial, accurately draw 0.5 mL of soil suspension using a clean 2.0 mL syringe and place it in a centrifuge tube containing 2.5 mL of 1.0 mol / L potassium chloride. Extract for 60 min by shaking at a constant temperature (180 rpm, 30℃) in the dark. After centrifugation, draw 0.2 mL of the supernatant and then determine the concentration using the indophenol blue colorimetric method. Calculate the actual content of both substances in the extracted sample based on the ammonium nitrogen standard.

[0107] The experimental steps are as follows:

[0108] 1) Add 50 mL of soil simulation solution to a 100 mL vial to construct a microcosm culture system; the soil simulation solution includes: anaerobic borax-boric acid buffer, nitrate, and sodium lactate. The concentration of borax-boric acid buffer is 50 mmol / L, pH = 7.0, the concentration of nitrate is 5.0 ± 0.2 mmol / L, and the concentration of sodium lactate is 1.2 mmol / L.

[0109] 2) Two experimental groups were set up, with 3 replicates in each group. Experimental group 1 was supplemented with 1.5g of the porous composite bacterial agent prepared in Example 1, and experimental group 2 was supplemented with 20mL of OD... 600 =1.25 KP bacterial solution; after adding the porous compound bacterial agent and KP bacterial solution, seal the vial, shake well, and then place all the vials in a constant temperature incubator and incubate at 28℃ for 7 days. Measure the changes in the concentrations of nitrate, nitrite and ammonium nitrogen in the reaction system every 12 hours.

[0110] Figure 1 This is a schematic diagram illustrating the change in nitrate content in Experiment Example 1. Figure 1 It can be seen that within 0-7 days, the nitrate concentration in both experimental groups 1 and 2 showed a decreasing trend, indicating that the addition of the porous composite bacterial agent and KP bacterial solution prepared in Example 1 can achieve the purpose of nitrate reduction. However, compared with experimental group 2, the nitrate concentration in experimental group 1 began to decrease sharply from the first day of static culture. By day 2.5, the nitrate concentration of 5.2 mmol / L in the system had been completely reacted, while in experimental group 2, the nitrate concentration in the system only decreased from 5.1 mmol / L to 0.8 mmol / L on day 2.5, and the reaction was not yet complete. The rate of nitrate reduction in experimental group 1 with the addition of porous composite bacterial agent was 1.27 times higher than that in the control group, indicating that compared with a single microorganism with nitrate reduction ability, the porous composite bacterial agent provided by this invention can significantly promote the nitrate reduction process.

[0111] Figure 2 This is a schematic diagram illustrating the change in nitrite content in Experiment Example 1. Figure 2 It can be seen that nitrite, as a reduction product of nitrate, showed significant differences in its variation among different treatment groups. In experimental group 1, the nitrite content peaked on day 2.5 of the reaction and showed a decreasing trend from day 2.5 to day 7. On day 4 of the reaction, the nitrite concentration decreased to 2.8 mmol / L. In experimental group 2, the nitrite concentration increased with the increase of culture time, reaching 3.7 mmol / L on day 4 of culture, and then gradually decreased. Compared with experimental group 2, which only added KP bacterial solution, experimental group 1, which added porous composite bacterial agent, significantly shortened the nitrite accumulation time and improved the nitrite degradation rate.

[0112] Figure 3 This is a schematic diagram illustrating the change in ammonium nitrogen content in Experiment Example 1. Figure 3It was observed that within 0-7 days, the concentration of ammonium nitrogen in both experimental groups 1 and 2 showed an increasing trend. However, in experimental group 1, which added porous composite bacterial agent, the accumulation rate of ammonium nitrogen was significantly accelerated, reaching 4.5 mmol / L after 4 days of reaction. In contrast, in experimental group 2, which only added KP bacterial solution, the accumulation rate of ammonium nitrogen was relatively slow, reaching only 3.1 mmol / L after 4 days of reaction. The efficiency of dissimilatory nitrate to ammonium production was calculated by measuring the decrease in nitrate and the amount of ammonium nitrogen produced. It was found that the efficiency in experimental group 2, which only added KP bacterial solution, was only 38%, while in experimental group 1, which added porous composite bacterial agent, the efficiency of dissimilatory nitrate to ammonium production increased to 52%. This indicates that the porous composite bacterial agent in this invention promotes both the reduction rate of nitrate and the conversion efficiency of dissimilatory nitrate.

[0113] Experimental Example 2: Nitrite Conversion Experiment

[0114] Test method:

[0115] Add 50-80 mL of soil simulation solution to 100-150 mL vials to construct a microcosm culture system. The soil simulation solution includes: anaerobic borax-boric acid buffer, nitrate, and sodium lactate. The concentration of the borax-boric acid buffer is 30 mmol / L, pH = 6.8-7.0, the concentration of nitrite is 1.8-2.7 mmol / L, and the concentration of sodium lactate is 0.8-1.3 mmol / L. The experiment is divided into two groups: one is the experimental group, which adds 1-2 g of the synthesized porous composite bacterial agent; the other is the control group, which adds only KP bacterial solution. After adding the porous composite bacterial agent and bacterial solution, the vials are sealed and shaken well. Then, three vials from each group are placed in a constant temperature incubator and statically cultured at 28-30℃. The concentration changes of nitrite, nitrous oxide, and ammonium nitrogen in the reaction system are measured every 12 hours.

[0116] The methods for determining the concentrations of nitrite and ammonium nitrogen are the same as in Experiment 1.

[0117] The method for determining nitrous oxide concentration is as follows: Nitrous oxide gas samples are tested before liquid sample collection. Before sampling, the vial is shaken well and allowed to stand in the dark for 15 minutes; a clean 1mL syringe is taken, and after cleaning the syringe with high-purity nitrogen, 0.8mL of headspace gas is drawn from the vial; after sampling, the sample is measured using a gas chromatograph, and the final content is determined according to the nitrous oxide standard.

[0118] The experimental steps are as follows:

[0119] 1) Add 50 mL of soil simulation solution to a 100 mL vial to construct a microcosm culture system; the soil simulation solution includes: anaerobic borax-boric acid buffer, nitrate, and sodium lactate. The concentration of borax-boric acid buffer is 30 mmol / L, pH = 7.0, the concentration of nitrate is 3.6 ± 0.2 mmol / L, and the concentration of sodium lactate is 1.0 mmol / L.

[0120] 2) Two experimental groups were set up, with 3 replicates in each group. 1g of the porous composite bacterial agent prepared in Example 2 was added to experimental group 1, and 20mL of OD200 was added to experimental group 2. 600 =1.25 KP bacterial solution; after adding porous composite bacterial agent and KP bacterial solution, seal the vial, shake well, and then place all vials in a constant temperature incubator and incubate at 28℃ for 6 days. The concentration changes of nitrite, nitrous oxide and ammonium nitrogen in the reaction system are measured every 12 hours.

[0121] Figure 4 This is a schematic diagram illustrating the change in nitrite content in Experiment Example 2. Figure 4 It can be seen that the concentration of nitrite in both experimental groups 1 and 2 showed a decreasing trend, indicating that nitrite underwent dissimilatory reduction. In experimental group 1, which added the porous composite bacterial agent prepared in Example 2, the added 3.7 mmol / L nitrate was completely reacted after 2.5 days of reaction. In experimental group 2, which added KP bacterial solution, the concentration of nitrite only decreased from 3.8 mmol / L to 0.6 mmol / L after 3 days of reaction, indicating that the reaction was not yet complete. Within the 2.5-day reaction period, the rate of decrease of nitrite content in experimental group 1 with the porous composite bacterial agent was 1.43 times higher than that in experimental group 2, indicating that the porous composite bacterial agent provided by the present invention is beneficial to promoting the dissimilatory reduction process of nitrite.

[0122] Figure 5 This is a schematic diagram illustrating the change in nitrous oxide content in Experiment Example 2. Figure 5 It can be seen that nitrous oxide, as a reduction product of nitrite, exhibits significant differences in its variation across different treatment groups. In experimental group 1, the nitrous oxide content reached 0.11 mmol / L after 2 days of reaction, and then slowly increased, reaching 0.30 mmol / L after 4 days of reaction. In experimental group 2, the nitrous oxide concentration increased with increasing culture time, reaching 0.15 mmol / L after 2 days of culture and 0.42 mmol / L after 6 days of reaction. During the reaction process of 1.8-6 days, the nitrous oxide concentration in experimental group 2 was consistently higher than that in experimental group 1, indicating that compared to a single microorganism with nitrate-reducing capabilities, the porous composite bacterial agent provided by this invention can reduce the production of nitrous oxide, which is beneficial for greenhouse gas emission reduction.

[0123] Figure 6 This is a schematic diagram illustrating the change in ammonium nitrogen content in Experiment Example 2. Figure 6 It can be seen that the concentration of ammonium nitrogen in both experimental groups 1 and 2 showed an increasing trend. In experimental group 1, which added the porous composite bacterial agent prepared in Example 2, the accumulation rate of ammonium nitrogen was significantly faster, reaching 3.1 mmol / L after 6 days of reaction. In contrast, in experimental group 2, which only added KP bacterial solution, the accumulation rate of ammonium nitrogen was relatively slow, reaching only 2.4 mmol / L after 6 days of reaction. The efficiency of ammonium production was calculated by measuring the decrease in nitrite and the amount of ammonium nitrogen produced. It was found that the efficiency in experimental group 2, which only added KP bacterial solution, was only 64%, while in experimental group 1, which added the porous composite bacterial agent, the efficiency increased to 83%. This indicates that the porous composite bacterial agent provided by this invention promotes both the reduction rate of nitrite and the conversion efficiency of dissimilatory nitrate. Example 3: Soil Nitrate Reduction Regulation Experiment

[0124] Test method:

[0125] Soil samples were collected for testing, and basic physicochemical properties of the soil were measured, including soil pH, total iron content, organic nitrogen content, and cation exchange capacity. After collection, soil samples were air-dried indoors, impurities such as stones were removed, and the samples were passed through a 10-mesh sieve for later use. Nine 5.0g soil samples were weighed and added to nine 150mL vials, with 80mL of deionized water added to each. The nine vials were divided into experimental groups 1-3 (three vials per group). Experimental group 1 was the blank group, with no added materials; experimental group 2 was the pure bacterial treatment group, with 0.5mL of OD245 added. 600 =1.0 KP bacterial suspension, and 1 mL each of 0.4 mol / L sodium nitrate and sodium acetate solutions. Experimental group 3 was a porous composite bacterial agent treatment group, with 0.5 g of porous composite bacterial agent, and 1 mL each of 0.4 mol / L sodium nitrate and sodium acetate solutions added. After the materials were added, the vials were sealed and shaken well. Then, the three vials (three of each group) were placed in a constant temperature incubator and statically cultured at 28-30℃. The concentration changes of nitrate, ammonium nitrogen, and dissolved organic carbon in the reaction system were measured every 12 hours.

[0126] The methods for determining the concentrations of nitrate and ammonium nitrogen are the same as in Experiment 1.

[0127] The method for determining the concentration of dissolved organic carbon is as follows: take a soil sample pore water solution, filter it through a 0.45 μm filter membrane, and then use a total organic carbon analyzer to determine the content of dissolved organic carbon (DOC) in the soil.

[0128] The experimental steps are as follows:

[0129] 1) Paddy soil from Zhishan Town, Jiangmen City, Guangdong Province was selected as the test soil sample. The basic physicochemical properties of the soil are shown in Table 1. After collection, the soil samples were naturally dried indoors, and impurities such as stones were removed. The samples were then passed through a 10-mesh sieve for later use.

[0130] 2) Weigh nine 5.0g soil samples and add them to nine 150mL vials, then add 80mL of deionized water to each vial. Divide the nine vials into experimental groups 1-3 (three vials in each group). Experimental group 1 is the blank group, with no added materials. Experimental group 2 is the pure bacterial treatment group, with 0.5mL of OD200 added. 600 The test group consisted of 1 mL of KP bacterial suspension (1.0 g), 1 mL of 0.4 mol / L sodium nitrate solution, and 1 mL of 0.4 mol / L sodium acetate solution. Group 3 was a porous composite bacterial agent treatment group, which included 0.5 g of the porous composite bacterial agent prepared in Example 3, along with 1 mL of 0.4 mol / L sodium nitrate solution and 1 mL of 0.4 mol / L sodium acetate solution. After adding the materials, the vials were sealed and shaken well. Then, the three vials (three of each group) were placed in a constant temperature incubator and incubated at 28°C for 5 days. The concentrations of nitrate, ammonium nitrogen, and dissolved organic carbon in the reaction system were measured every 12 hours.

[0131] Table 1. Basic physicochemical properties of soil samples from Experiment Example 3

[0132] Soil location Guangdong Zhishan pH 5.47 Total iron content (g / kg) 19.7 Organic nitrogen content (g / kg) 26.9 Cation exchange capacity (cmol / kg) 8.34

[0133] Figure 7 This is a schematic diagram illustrating the change in nitrate content in Experiment Example 3. Figure 7 It was observed that the nitrate reduction rate was very slow in experimental group 1. In experimental groups 2 and 3, which were respectively supplemented with KP bacterial solution and the porous composite bacterial agent prepared in Example 3, the nitrate concentration decreased rapidly, indicating that the nitrate was reduced. After 5 days of reaction, the nitrate concentration in experimental groups 1-3 decreased from 5.6 mmol / L to 3.8 mmol / L, 1.9 mmol / L, and 0.4 mmol / L, respectively. Compared with experimental group 1, the nitrate degradation in experimental groups 2 and 3 increased by 2.05 and 2.89 times, respectively, indicating that the porous composite bacterial agent has a more significant effect in promoting the reduction of soil nitrate.

[0134] Figure 8 This is a schematic diagram illustrating the change in ammonium nitrogen content in Experiment Example 3. Figure 8It can be seen that the initial concentration of ammonium nitrogen was the same in different treatment groups, which may be due to the background value of the soil, and the concentration of ammonium nitrogen gradually increased with the incubation time. In experimental group 1, the increase in ammonium nitrogen was very low, with an accumulation of only 0.8 mmol / L after 5 days of reaction. In experimental group 2, which only added KP bacterial solution, ammonium nitrogen was gradually produced and accumulated, reaching an accumulation of 2.9 mmol / L after 5 days of reaction. In experimental group 3, which added the porous composite bacterial agent prepared in Example 3, the accumulation rate of ammonium nitrogen was significantly accelerated, reaching an accumulation of 3.9 mmol / L after 5 days of reaction. The efficiency of ammonium production was calculated by the decrease in nitrate and the amount of ammonium nitrogen produced. The efficiencies of experimental groups 2 and 3 were 54% and 70%, respectively, indicating that the porous composite bacterial agent provided by the present invention promoted both the reduction rate of nitrate and the conversion efficiency of dissimilatory nitrate.

[0135] Figure 9 This is a schematic diagram illustrating the change in dissolved organic carbon content in Experimental Example 3. Figure 9 It can be seen that the concentration of dissolved organic carbon differed among the different treatment groups after the reaction. In experimental groups 1-3, the concentrations of dissolved organic carbon were 30 mg / L, 34 mg / L, and 42 mg / L, respectively, indicating that the porous composite bacterial agent provided by this invention also has the function of increasing the organic carbon content in the soil system.

Claims

1. The application of porous composite microbial agents in enhancing the dissimilatory reduction of soil nitrates to ammonium, characterized in that, The porous composite bacterial agent is a modified porous mineral loaded with microorganisms capable of reducing nitrates; The microorganisms with nitrate-reducing ability are selected from... Klebsiella pneumoniae ; The modified porous mineral is prepared from the following raw materials: natural porous mineral, crop straw, active organic matter, composite fiber and water; the active organic matter is selected from xylitol; the composite fiber is selected from lignin-based carbon fiber.

2. The application according to claim 1, characterized in that, The particle size of the natural porous mineral is 0.5-2 mm; And / or, the specific surface area of ​​the natural porous mineral is 2-5 m². 2 / g; And / or, the particle size of the crop straw is less than or equal to 2 mm; And / or, the specific surface area of ​​the modified porous mineral is 10-20 m². 2 / g.

3. The application according to claim 1, characterized in that, The natural porous mineral is selected from at least one of perlite, vermiculite, diatomite, volcanic rock, maifanite, and zeolite.

4. The application according to claim 1, characterized in that, The modified porous mineral is prepared by a method comprising the following steps: Natural porous minerals and crop straw are mixed and then pyrolyzed and carbonized to obtain porous minerals with a biochar layer. The active organic matter, composite fiber and water are mixed, and the porous mineral with the biochar layer is added and dried to obtain the modified porous mineral.

5. The application according to claim 4, characterized in that, The mass ratio of the natural porous mineral to crop straw is (5-25):1; And / or, the heating program for the pyrolysis carbonization is as follows: heating at 120-170℃ for 30-60 min, and then heating to 700-850℃ for 30-60 s; And / or, the mass ratio of the active organic matter, composite fiber, water and porous mineral with biochar layer is 1: (5-15): (60-110): (500-850).

6. The application according to any one of claims 1-5, characterized in that, The porous composite bacterial agent is prepared by a method comprising the following steps: The modified porous minerals were co-cultured with microorganisms capable of reducing nitrates to obtain the porous composite bacterial agent.

7. The application according to claim 6, characterized in that, The density of the microorganisms with nitrate-reducing ability is OD. 600 =1-1.5; the solid-liquid ratio of the porous mineral to the microorganism with nitrate-reducing ability is 1g: (0.2-1)mL; And / or, the co-culture temperature is 25-30℃ and the time is 15-20h.

8. The application according to claim 1, characterized in that, The mass ratio of the porous composite microbial agent to the soil is 1:(10-20); And / or, the physical and chemical properties of the soil include at least one of the following: pH value is 4.0-8.0; The total iron content is 15-30 g / kg; The organic nitrogen content is 20-35 g / kg; The cation exchange capacity is 5-15 cmol / kg.

Images