Porous complex microbial inoculant for promoting nitrate reduction to produce ammonium as well as preparation method and application of porous complex microbial inoculant

Abstract

The invention relates to the technical field of soil improvement, and discloses a porous complex microbial inoculant for promoting nitrate reduction to produce ammonium as well as a preparation method and application of the porous complex microbial inoculant. The invention relates to a porous complex microbial inoculant which is a modified porous mineral loaded with microorganisms with nitrate reducing capacity, the microorganism with the nitrate reducing capacity is prepared from at least one of Klebsiella pneumoniae, pseudomonas nitritiducen and Shewanella pneumoniae strain CN32, and the microorganism with the nitrate reducing capacity is prepared from at least one of Klebsiella pneumoniae, pseudomonas nitritiducen and Shewanella pneumoniae strain CN32. The porous complex microbial inoculant provided by the invention can promote the efficiency and rate of conversion from nitrate or nitrite to ammonium nitrogen, reduce the loss of nitrogen and the generation of greenhouse gases, has a compact structure and good stability, and can continuously play a role; the method is applied to the process of strengthening dissimilatory reduction of soil nitrate into ammonium, and soil nutrient loss and air pollution can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of soil improvement, and particularly relates to a porous composite microbial agent for promoting ammonium production by nitrate reduction, a preparation method thereof, and an application thereof. Background Art

[0002] The application of nitrogen fertilizers with nitrate nitrogen and ammonium nitrogen as the core plays an important role in ensuring food production. Compared with ammonium nitrogen, nitrate nitrogen is easily transported with water bodies and produces gaseous nitrogen products, which easily causes nutrient loss in farmland soil and environmental problems.

[0003] The conversion of nitrate nitrogen in agricultural soil mainly includes two processes: denitrification and dissimilatory nitrate reduction. Among them, the denitrification process will produce nitrogen gas and the greenhouse gas nitrous oxide, causing environmental problems such as nitrogen loss and air pollution. Dissimilatory nitrate reduction (i.e., the DNRA process) can ensure the storage of nitrogen in the soil by converting nitrate into ammonium nitrogen. However, the denitrification process dominates in the soil. Therefore, improving the role of dissimilatory nitrate reduction in the soil and promoting the conversion of soil nitrate into ammonium nitrogen are of great significance for improving the utilization efficiency of soil fertilizer, reducing nitrogen pollution, and developing high-quality agricultural construction.

[0004] CN 115974288 A discloses a method for enhancing the dissimilatory reduction of nitrate to ammonium by coupling an electric field and a magnetic field. The coupling effect of the electric field and the magnetic field promotes the rapid startup of the DNRA reactor and improves the stability of the system. At the same time, the activity of DNRA bacteria and the abundance of the functional gene nrfA are improved. However, this method is only applicable to the water environment, is difficult to be popularized and play a role in the regulation of farmland soil, and requires continuous power consumption, with high costs. CN 117070415 A discloses a highly efficient denitrifying strain with the function of dissimilatory reduction to ammonium and its application. This strain can rapidly remove nitrate nitrogen in the water body while effectively reducing the generation of nitrogen oxide gases. However, the use of this strain is still limited to the water environment and does not involve the improvement of soil nitrogen transformation.

[0005] It can be seen that the current research on strengthening the DNRA process mainly focuses on the water treatment system, and the methods mainly include electrochemical strengthening and screening of new strains. For the method of strengthening the DNRA process in soil, further research is still needed. Summary of the Invention

[0006] The present invention aims to solve at least one of the above technical problems existing in the prior art. For this reason, one of the purposes of the present invention is to provide a porous composite microbial agent.

[0007] The second purpose of the present invention is to provide a preparation method of this porous composite microbial agent.

[0008] A third object of the present invention is to provide the application of such a porous composite microbial agent.

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

[0010] The first aspect of the present invention provides a porous composite microbial agent, and the porous composite microbial agent is a modified porous mineral loaded with microorganisms having nitrate reduction ability;

[0011] Among them, the microorganisms having nitrate reduction 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 (abbreviated as KP) and pseudomonas nitritireducen are purchased from the China Center for Marine Microbial Culture Collection Management, and the preservation numbers are 1A18916 and 1A12677 respectively; Shewanella putrefaciens strain CN32 (abbreviated as CN32) is purchased from the Guangdong Provincial Microbial Culture Collection Center, and the preservation number is 1.459.

[0013] In some embodiments of the present invention, the modified porous mineral is prepared from the following raw materials: natural porous mineral, crop straw, active organic matter, composite fiber 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 m 2 / g.

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

[0021] In some specific embodiments of the present invention, the specific surface area of the modified porous mineral is 15-20 m 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 stone, medical stone, 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 the present invention, substances rich in hydroxyl groups such as xylitol, maltitol, and sorbitol are selected as the active organic matter. The hydroxyl functional groups therein can form intermolecular hydrogen bonds with the hydroxyl, carboxyl and other active groups on the surface of the composite fiber, the microbial cells with nitrate reduction ability, and the surface of the modified porous mineral, so that the raw materials for preparing the porous composite bactericide are firmly combined together, thereby obtaining a porous composite bactericide 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 the present invention, cereal crop straws containing rich cellulose, hemicellulose and lignin are selected as the raw materials for preparing the modified porous mineral. In the subsequent pyrolysis carbonization process, it is more conducive to forming 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] Mix the natural porous mineral and the crop straw, and pyrolyze and carbonize to obtain a porous mineral with a biochar layer;

[0031] Mix the active organic matter, the composite fiber and water, add the porous mineral with the biochar layer, and dry to obtain the modified porous mineral.

[0032] In some embodiments of the present invention, after adding the porous mineral with a biochar layer, it further includes stirring and mixing for 20 - 40 min.

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

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

[0035] In some embodiments of the present invention, the heating program for pyrolytic carbonization is: heating at 120 - 170 °C for 30 - 60 min, and then raising the temperature to 700 - 850 °C and heating for 30 - 60 s.

[0036] In some specific embodiments of the present invention, the heating program for pyrolytic carbonization is: heating at 130 - 160 °C for 30 - 40 min, and then raising the temperature to 700 - 800 °C and heating for 30 - 50 s.

[0037] In some embodiments of the present invention, the mass ratio of the active organic matter, composite fiber, water, and the porous mineral with a 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 the porous mineral with a 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 h.

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

[0041] The second aspect of the present invention provides a preparation method of the porous composite bacterium agent described in the first aspect of the present invention, including the following steps:

[0042] Co - culture the modified porous mineral with the microorganism having nitrate reduction ability to obtain the porous composite bacterium agent.

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

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

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

[0046] In some specific embodiments of the present invention, the buffer solution includes a boric acid-borax buffer solution.

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

[0048] In some specific embodiments of the present invention, the nutrient component includes sodium nitrate and sodium lactate with a mass ratio of 1 : (1 - 2).

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

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

[0051] In some embodiments of the present invention, after the co-culture, it further includes steps of centrifugation, solid-liquid separation to collect the solid phase to obtain the porous composite microbial agent.

[0052] The third aspect of the present invention provides the application of the porous composite microbial agent described in the first aspect of the present invention in enhancing the process of soil nitrate dissimilatory reduction 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 physical and chemical properties of the soil include at least one of the following:

[0056] The 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 physical and chemical properties of the soil include at least one of the following:

[0061] The 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 principle of the present invention is described as follows:

[0066] 1) First, the present invention uses natural porous minerals and crop straws as raw materials, and through pyrolytic carbonization, porous minerals with a biochar layer are obtained. The porous minerals with a biochar layer have a rich pore structure and a large specific surface area. On the one hand, the porous structure provides a large number of attachment sites for microorganisms with nitrate reduction ability, enabling the microorganisms to stably exist in the microbial agent and not easily flow away; 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 opportunity between the microorganisms and the substrate, thereby promoting the conversion of nitrates; at the same time, this mineral-biochar composite structure can also improve the aeration and water retention of the soil, increase soil voids and respiration ability, and create a suitable living environment for microorganisms;

[0067] 2) After mixing the porous mineral with a biochar layer, active organic matter, composite fibers, and water, ① the composite fibers have high strength and stability and can be interconnected with raw materials such as the porous mineral with a biochar layer and active organic matter through chemical bonds (such as covalent bonds, hydrogen bonds, and π-π stacking interactions), enhancing the structural stability of the entire composite microbial agent and enabling it to remain stable in the soil environment for a long time. In addition, carbon fibers have good electrical conductivity and can promote electron transfer during the metabolism of microorganisms. Electron transfer plays a key role in the process of nitrate reduction to ammonium nitrogen. Composite fibers such as lignin-based carbon fibers can accelerate the electron transfer rate, improve the efficiency of the nitrate reduction reaction, and thus promote the DNRA process and increase the generation of ammonium nitrogen. ② Active organic matter such as xylitol is an excellent carbon source for the growth and metabolism of microorganisms with nitrate reduction ability. In the soil environment, microorganisms use active organic matter for growth and reproduction, providing the energy and material basis for the nitrate reduction process. At the same time, the presence of active organic matter can also regulate the metabolic pathways of microorganisms. Research shows that appropriate carbon sources can affect the metabolic direction of microorganisms, making nitrate-reducing bacteria more inclined to reduce nitrate to ammonium nitrogen through the DNRA pathway rather than denitrification to produce gases such as nitrogen and nitrous oxide, thereby reducing nitrogen loss caused by the denitrification process.

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

[0069] 4) The porous composite microbial agent also contains abundant organic matter. For example, biochar directly supplements the organic carbon source for the soil, increasing the content of soil organic matter. Part of the active organic matter is 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 the soil properties in the soil.

[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 the conversion of nitrate or nitrite to ammonium nitrogen, reduce nitrogen loss and greenhouse gas generation. The porous composite microbial agent has a compact structure and good stability and can continuously play a role.

[0072] 2) The preparation method of the porous composite bacterial agent provided by the present invention has simple steps, low raw material cost, does not require the use of complex instruments, does not generate waste, is environmentally friendly, and is suitable for industrial application;

[0073] 3) The porous composite bacterial agent provided by the present invention can be applied to enhance the process of nitrate dissimilatory reduction to ammonium in soil, reduce soil nutrient loss and air pollution. When applied to soil, it can also increase soil porosity and respiration ability, increase soil organic carbon content, and improve soil properties. Description of the Drawings

[0074] Figure 1 It is a schematic diagram of the content change of nitrate in Test Example 1;

[0075] Figure 2 It is a schematic diagram of the content change of nitrite in Test Example 1;

[0076] Figure 3 It is a schematic diagram of the content change of ammonium nitrogen in Test Example 1;

[0077] Figure 4 It is a schematic diagram of the content change of nitrite in Test Example 2;

[0078] Figure 5 It is a schematic diagram of the content change of nitrous oxide in Test Example 2;

[0079] Figure 6 It is a schematic diagram of the content change of ammonium nitrogen in Test Example 2;

[0080] Figure 7 It is a schematic diagram of the content change of nitrate in Test Example 3;

[0081] Figure 8 It is a schematic diagram of the content change of ammonium nitrogen in Test Example 3;

[0082] Figure 9 It is a schematic diagram of the content change of dissolved organic carbon in Test Example 3. Detailed Embodiments

[0083] The content of the present invention will be further described in detail through specific embodiments below. The raw materials, reagents or devices used in the embodiments and test examples can be obtained from conventional commercial channels or can be obtained by existing technical methods unless otherwise specified. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0084] Example 1

[0085] In this example, a porous composite bacterial agent is prepared, and the steps are as follows:

[0086] S11. Take particles with a particle size of 0.5 - 1 mm and a specific surface area of 2 - 4 m 21 kg of perlite with a particle size of 0.5 - 2 mm and a specific surface area of 2 - 3 m² / g is placed in a cool and 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 a powder with a particle size of 0.15 - 2 mm using a pulverizer;

[0087] S12: After evenly mixing the treated perlite and the rice straw powder, put them into a muffle furnace (where the mass ratio of perlite to rice straw powder is 13:1). Start the heating program. First, heat at 150 °C for 35 min, then raise the temperature to 800 °C and continue heating for 40 s to obtain a porous mineral with a biochar layer;

[0088] S21: Accurately weigh 0.5 g of xylitol and 5 g of lignin-based carbon fiber and place them in a 1.5 L aluminum box. Add 50 mL of deionized water, mix evenly, then add 400 g of the porous mineral with a biochar layer. Seal the aluminum box, stir and turn it for 30 min, and then place the sample in an oven at 40 °C for 12 h to obtain a modified porous mineral;

[0089] S31: Add 500 mL of an anaerobic and sterile 50 mmol / L boric acid - borax buffer solution (pH = 7.0), 50 g of the modified porous mineral, 20 mL of a KP bacterial solution with an OD 600 = 1.25, 0.3 g of sodium nitrate, and 0.5 g of sodium lactate to a large Erlenmeyer flask. After shaking well, place the Erlenmeyer flask in an incubator and statically culture at 28 °C for 18 h. After the culture is completed, centrifuge the sample and collect the precipitate to obtain a porous composite bacterial agent with a specific surface area of 16.3 m 2 ² / g.

[0090] Example 2

[0091] To prepare a porous composite bacterial agent in this example, the steps are as follows:

[0092] S11: Take 1 kg of perlite with a particle size of 0.5 - 2 mm and a specific surface area of 2 - 3 m² / g, and dry it 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 a powder with a particle size of 0.15 - 2 mm using a pulverizer; 2 1 kg of perlite with a particle size of 0.5 - 2 mm and a specific surface area of 2 - 3 m² / g is placed in a cool and 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 a powder with a particle size of 0.15 - 2 mm using a pulverizer;

[0093] S12: After evenly mixing the treated perlite and the rice straw powder, put them into a muffle furnace (where the mass ratio of perlite to rice straw powder is 19:1). Start the heating program. First, heat at 160 °C for 35 min, then raise the temperature to 800 °C and continue heating for 45 s to obtain a porous mineral with a biochar layer;

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

[0095] S31. Add 500 mL of anaerobic and sterile 50 mmol / L boric acid borax buffer solution (pH = 7.0), 50 g of modified porous mineral, and 20 mL of OD 600 = 1.25 KP bacterial solution, 0.3g sodium nitrate and 0.5g sodium lactate, shake well and put the vial into the incubator, incubate at 28℃ for 18h, centrifuge the sample after incubation, collect the precipitate, and obtain a specific surface area of ​​15.4m 2 / g of porous composite bacterial agent.

[0096] Example 3

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

[0098] S11, particle size 0.5-1.5mm, specific surface area 2-3m 2 1 kg of perlite with a particle size of 0.15-2 mm is dried in the sun 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 a powder with a crusher with a particle size of 0.15-2 mm.

[0099] S12, the treated perlite and rice straw powder are evenly mixed and put into a muffle furnace (wherein the mass ratio of perlite to rice straw powder is 7:1), and the heating program is started. After heating at 130° C. for 35 minutes, the temperature is increased to 700° C. and continued to be heated for 45 seconds to obtain a porous mineral with a biochar layer;

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

[0101] S31. Add 500 mL of anaerobic and sterile 50 mmol / L boric acid borax buffer solution (pH = 7.0), 50 g of modified porous mineral, and 20 mL of OD 600 = 1.25 KP bacterial solution, 0.3g sodium nitrate and 0.5g sodium lactate, shake well and put the vial into the incubator, incubate at 28℃ for 18h, centrifuge the sample after incubation, collect the precipitate, and obtain a specific surface area of ​​15.8m 2The porous composite microbial agent of / g.

[0102] Experimental Example 1 Nitrate Conversion Experiment

[0103] Test method:

[0104] Add 50 - 80 mL of soil simulation solution to a 100 - 150 mL vial to construct a microcosm culture system. The soil simulation solution includes: anaerobic borax - boric acid buffer, nitrate, and sodium lactate. Among them, 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 group is the experimental group, which adds 1 - 2 g of the synthesized porous composite microbial agent; the other group is the control group, which only adds KP bacterial solution. After adding the porous composite microbial agent and the bacterial solution, seal the vial, shake well, and then place the two groups of vials (three each) in a constant temperature incubator for static culture at 28 - 30 °C. Measure the concentration changes of nitrate, nitrite, and ammonium nitrogen in the reaction system every about 12 h.

[0105] Among them, the measurement method for the concentrations of nitrate and nitrite is as follows: Mix the samples in the vial, use a clean 5 mL syringe to suck 1 mL of the suspension into a centrifuge tube containing 4 mL of deionized water, extract on a shaker for 5 min, and the extraction parameters are light avoidance, 30 °C, and 200 rpm; after extraction, centrifuge the centrifuge tube for 3 min, and the centrifugation parameters are 25 °C and 6000 rpm; after centrifugation, take the supernatant and filter it through a 0.22 μm filter head, and then use an ion chromatograph to measure nitrate and nitrite, and calculate the actual contents of the two in the extracted sample based on the nitrate and nitrite calibration curves.

[0106] The measurement method for the concentration of ammonium nitrogen is as follows: Mix the samples in the vial, use a clean 2.0 mL syringe to accurately suck 0.5 mL of the soil suspension and place it in a centrifuge tube containing 2.5 mL of 1.0 mol / L potassium chloride, extract by shaking in the dark at a constant temperature (180 rpm, 30 °C) for 60 min, then centrifuge and suck 0.2 mL of the supernatant, and then use the indophenol blue colorimetric method for measurement, and calculate the actual content of the two in the extracted sample based on the ammonium nitrogen calibration curve.

[0107] The test 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. Among them, the concentration of the 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) Set up two experimental groups, with 3 parallel samples in each group. Add 1.5 g of the porous composite bacterial agent prepared in Example 1 to Experimental Group 1, and add 20 mL of KP bacterial solution with an OD 600 = 1.25 to Experimental Group 2; after adding the porous composite bacterial agent and the KP bacterial solution, seal the vials, shake well, and then place all the vials in a constant temperature incubator and statically culture at 28 °C for 7 d. Measure the concentration changes of nitrate, nitrite, and ammonium nitrogen in the reaction system every 12 h.

[0110] Figure 1 It is a schematic diagram of the content change of nitrate in Test Example 1. From Figure 1 it can be seen that within 0 - 7 d, the concentrations of nitrate in both Experimental Group 1 and Experimental Group 2 showed a downward trend, indicating that adding the porous composite bacterial agent prepared in Example 1 and the KP bacterial solution can both achieve the purpose of reducing nitrate. However, compared with Experimental Group 2, starting from the 1st d of static culture, the concentration of nitrate in Experimental Group 1 began to decrease sharply. At the 2.5th d of the system, 5.2 mmol / L of nitrate had been completely reacted, while in Experimental Group 2 at the 2.5th d, the concentration of nitrate in the system only decreased from 5.1 mmol / L to 0.8 mmol / L and had not reacted completely. The nitrate decline rate in Experimental Group 1 with the added porous composite bacterial agent was increased by 1.27 times compared with the control group, indicating that compared with a single microorganism with nitrate reduction ability, the porous composite bacterial agent provided by the present invention can significantly promote the nitrate reduction process.

[0111] Figure 2 It is a schematic diagram of the content change of nitrite in Test Example 1. From Figure 2 it can be seen that as the reduction product of nitrate, the changes of nitrite in different treatment groups showed great differences. In Experimental Group 1, the content of nitrite reached the peak at the 2.5th d of the reaction and showed a downward trend from the 2.5th d to the 7th d. At the 4th d of the reaction, the concentration of nitrite decreased to 2.8 mmol / L; in Experimental Group 2, the concentration of nitrite increased with the increase of the culture time and increased to 3.7 mmol / L on the 4th day of culture and then gradually decreased; compared with Experimental Group 2 with only the added KP bacterial solution, Experimental Group 1 with the added porous composite bacterial agent significantly shortened the time of nitrite accumulation and increased the degradation rate of nitrite.

[0112] Figure 3 It is a schematic diagram of the content change of ammonium nitrogen in Test Example 1. From Figure 3It can be seen that within 0 - 7 days, the concentration of ammonium nitrogen in both experimental group 1 and experimental group 2 showed an upward trend. However, in experimental group 1 with the addition of the porous composite bacterium agent, the cumulative rate of ammonium nitrogen increased significantly. After 4 days of reaction, the cumulative amount of ammonium nitrogen reached 4.5 mmol / L. In experimental group 2 with only the addition of KP bacterial solution, the cumulative rate of ammonium nitrogen was relatively slow. After 4 days of reaction, the cumulative amount was only 3.1 mmol / L. By calculating the efficiency of dissimilatory nitrate reduction to ammonium based on the decrease in nitrate and the production of ammonium nitrogen, it was found that the efficiency in experimental group 2 with only the addition of KP bacterial solution was only 38%, while in experimental group 1 with the addition of the porous composite bacterium agent, the efficiency of dissimilatory nitrate reduction to ammonium increased to 52%. This indicates that the porous composite bacterium agent in the present invention promotes the reduction rate of nitrate on the one hand and also promotes the conversion efficiency of dissimilatory nitrate on the other hand.

[0113] Experimental Example 2 Nitrite Conversion Experiment

[0114] Test method:

[0115] Add 50 - 80 mL of soil simulated solution to a 100 - 150 mL vial to construct a microcosm culture system. The soil simulated solution includes: anaerobic borax - boric acid buffer solution, nitrate, and sodium lactate. Among them, the concentration of the borax - boric acid buffer solution 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 group is the experimental group, adding 1 - 2 g of the synthesized porous composite bacterium agent; the other group is the control group, adding only KP bacterial solution. After adding the porous composite bacterium agent and the bacterial solution, seal the vial, shake well, and then place the two groups of vials (three for each group) in a constant temperature incubator for static culture at 28 - 30 °C. Measure the concentration changes of nitrite, nitrous oxide, and ammonium nitrogen in the reaction system every about 12 h.

[0116] Among them, the measurement methods of nitrite and ammonium nitrogen concentrations are the same as those in Experimental Example 1.

[0117] The measurement method of nitrous oxide concentration is as follows: The nitrous oxide gas sample is tested before sampling the liquid sample. Before sampling, shake the vial well and let it stand in the dark for 15 min; take a clean 1 mL syringe, wash the syringe with high - purity nitrogen, and then aspirate 0.8 mL of the headspace gas in the vial; after sampling, the sample is measured using a gas chromatograph, and the final content is determined according to the nitrous oxide calibration curve.

[0118] Test procedure:

[0119] 1) Add 50 mL of soil-simulating solution to a 100 mL vial to construct a microcosm culture system. The soil-simulating solution includes: anaerobic borax-boric acid buffer, nitrate, and sodium lactate. Among them, the concentration of the 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) Set up two experimental groups, with 3 parallel samples in each group. Add 1 g of the porous composite bactericide prepared in Example 2 to Experimental Group 1, and add 20 mL of KP bacterial solution with OD 600 = 1.25 to Experimental Group 2. After adding the porous composite bactericide and KP bacterial solution, seal the vial, shake well, and then place all the vials in a constant temperature incubator and incubate statically at 28°C for 6 d. Measure the concentration changes of nitrite, nitrous oxide, and ammonium nitrogen in the reaction system every 12 h.

[0121] Figure 4 It is a schematic diagram of the change in the content of nitrite in Test Example 2. As can be seen from Figure 4 it, the concentrations of nitrite in both Experimental Group 1 and Experimental Group 2 showed a downward trend, indicating that dissimilatory reduction of nitrite occurred. In Experimental Group 1 where the porous composite bactericide prepared in Example 2 was added, after 2.5 d of reaction, the added 3.7 mmol / L of nitrate was completely reacted. While in Experimental Group 2 where KP bacterial solution was added, after 3 d of reaction, the concentration of nitrite only decreased from 3.8 mmol / L to 0.6 mmol / L and was not completely reacted. During the 2.5 d of reaction time, the decline rate of the nitrite content in Experimental Group 1 with the added porous composite bactericide was 1.43 times higher than that in Experimental Group 2, indicating that the porous composite bactericide provided by the present invention is beneficial to promoting the dissimilatory reduction process of nitrite.

[0122] Figure 5 It is a schematic diagram of the change in the content of nitrous oxide in Test Example 2. As can be seen from Figure 5 it, as a reduction product of nitrite, the change of nitrous oxide in different treatment groups showed great differences. In Experimental Group 1, the content of nitrous oxide reached 0.11 mmol / L when the reaction reached 2 d, and then increased slowly. After the reaction reached 4 d, the concentration of nitrous oxide increased to 0.30 mmol / L. In Experimental Group 2, the concentration of nitrous oxide increased with the increase of the culture time, increased to 0.15 mmol / L after 2 d of culture, and increased to 0.42 mmol / L after 6 d of reaction. During the reaction process from 1.8 - 6 d, the concentration of nitrous oxide in Experimental Group 2 was always higher than that in Experimental Group 1, indicating that compared with a single microorganism with nitrate reduction ability, the porous composite bactericide provided by the present invention can reduce the production of nitrous oxide and is beneficial to the reduction of greenhouse gas emissions.

[0123] Figure 6 Schematic diagram of the change in the ammonium nitrogen content in Test Example 2. As can be seen from Figure 6 it, the concentrations of ammonium nitrogen in Test Group 1 and Test Group 2 both showed an upward trend. In Test Group 1 with the addition of the porous composite microbial agent prepared in Example 2, the cumulative rate of ammonium nitrogen was significantly faster. After 6 days of reaction, the cumulative amount of ammonium nitrogen reached 3.1 mmol / L. While in Test Group 2 with only the addition of KP bacterial solution, the cumulative rate of ammonium nitrogen was relatively slow. After 6 days of reaction, the cumulative amount was only 2.4 mmol / L. By calculating the ammonium production efficiency based on the decrease in nitrite and the production of ammonium nitrogen, it was found that the efficiency in Test Group 2 with only the addition of KP bacterial solution was only 64%, while in Test Group 1 with the addition of the porous composite microbial agent, the efficiency increased to 83%, indicating that the porous composite microbial agent provided by the present invention promoted the reduction rate of nitrite on the one hand and also promoted the efficiency of dissimilatory nitrate conversion on the other hand. Test Example 3 Soil nitrate reduction regulation experiment

[0124] Test method:

[0125] Collect the soil samples to be tested, measure the basic physical and chemical properties of the soil, including soil pH, total iron content, organic nitrogen content, and cation exchange capacity. After the soil samples are collected, they are naturally dried indoors, stones and other impurities are picked out, and they are passed through a 10-mesh sieve for standby. Weigh 9 portions of 5.0 g soil samples and add them to 9 150 mL vials respectively, and add 80 mL of deionized water to each. Divide the 9 vials into Test Groups 1-3 (three vials in each group). Among them, Test Group 1 is the blank group without adding any materials, Test Group 2 is the pure bacteria treatment group, adding 0.5 mL of KP bacterial solution with OD 600 = 1.0, as well as 1 mL of 0.4 mol / L sodium nitrate and sodium acetate solutions each, and Test Group 3 is the porous composite microbial agent treatment group, adding 0.5 g of the porous composite microbial agent, as well as 1 mL of 0.4 mol / L sodium nitrate and sodium acetate solutions each. After adding the materials, seal the vials and shake well, then place the three groups of vials (three in each group) in a constant temperature incubator and statically culture at 28-30 °C, and measure the concentration changes of nitrate, ammonium nitrogen, and dissolved organic carbon in the reaction system every about 12 h.

[0126] Among them, the measurement methods of nitrate and ammonium nitrogen concentrations are the same as those in Test Example 1.

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

[0128] Test steps are as follows:

[0129] 1) Select the paddy soil in Zhishan Town, Jiangmen City, Guangdong Province as the tested soil sample. The basic physical and chemical property data of the soil are shown in Table 1. After the soil sample is collected, it is naturally dried indoors, stones and other impurities are picked out, and it is passed through a 10-mesh sieve for standby.

[0130] 2) Weigh 9 portions of 5.0 g soil samples and add them into 9 150 mL vials respectively, and then add 80 mL deionized water to each vial. Divide the 9 vials into experimental groups 1 - 3 (three vials in each group). Among them, experimental group 1 is the blank group without adding any materials; experimental group 2 is the pure bacteria treatment group, adding 0.5 mL of KP bacterial solution with OD 600 = 1.0, as well as 1 mL of 0.4 mol / L sodium nitrate and sodium acetate solutions each; experimental group 3 is the porous composite bacterium agent treatment group, adding 0.5 g of the porous composite bacterium agent prepared in Example 3, and 1 mL of 0.4 mol / L sodium nitrate and sodium acetate solutions each. After adding the materials, seal the vials and shake well, then place the three groups of vials (three in each group) in a constant temperature incubator and statically culture at 28 °C for 5 d. Measure the concentration changes of nitrate, ammonium nitrogen and dissolved organic carbon in the reaction system every 12 h.

[0131] Table 1 Basic physical and chemical properties of the soil sample in Test Example 3

[0132] Soil Location Zhishan, Guangdong 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 is the schematic diagram of the content change of nitrate in Test Example 3. It can be seen from Figure 7 that in experimental group 1, the reduction rate of nitrate is very slow. In experimental groups 2 and 3 with the addition of KP bacterial solution and the porous composite bacterium agent prepared in Example 3 respectively, the nitrate concentration drops rapidly, indicating that nitrate is reduced. After 5 d of reaction, the nitrate concentrations in experimental groups 1 - 3 decrease 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 degradation amounts of nitrate in experimental groups 2 and 3 are increased by 2.05 and 2.89 times respectively, indicating that the porous composite bacterium agent has a more obvious effect in promoting the reduction of soil nitrate.

[0134] Figure 8 is the schematic diagram of the content change of ammonium nitrogen in Test Example 3. It can be seen from Figure 8It can be seen that the initial concentrations of ammonium nitrogen in different treatment groups are the same, probably from the background value of the soil, and the concentration of ammonium nitrogen increases gradually with the incubation time. In experimental group 1, the increase in ammonium nitrogen is very low, and the accumulation amount after 5 days of reaction is only 0.8 mmol / L. In experimental group 2 with only the addition of KP bacterial solution, ammonium nitrogen is gradually produced and accumulated. After 5 days of reaction, the accumulation amount reaches 2.9 mmol / L. While in experimental group 3 with the addition of the porous composite bacterial agent prepared in Example 3, the accumulation rate of ammonium nitrogen is significantly accelerated. After 5 days of reaction, the accumulation amount of ammonium nitrogen reaches 3.9 mmol / L. By calculating the ammonium production efficiency based on the decrease in nitrate and the production amount of ammonium nitrogen, the efficiencies of experimental groups 2 and 3 are 54% and 70% respectively, indicating that the porous composite bacterial agent provided by the present invention promotes the reduction rate of nitrate on the one hand and also promotes the efficiency of dissimilatory nitrate transformation on the other hand.

[0135] Figure 9 It is a schematic diagram of the change in the content of dissolved organic carbon in Test Example 3. From Figure 9 it can be seen that after the reaction, there are differences in the concentrations of dissolved organic carbon in different treatment groups. In experimental groups 1 - 3, the concentrations of dissolved organic carbon are 30 mg / L, 34 mg / L, and 42 mg / L respectively, indicating that the porous composite bacterial agent provided by the present invention also has the function of increasing the content of organic carbon in the soil system.

Claims

1. A porous composite microbial agent, characterized in that, The porous composite microbial agent is a modified porous mineral loaded with microorganisms having nitrate reduction ability; Among them, the microorganisms having nitrate reduction ability include at least one of Klebsiella pneumoniae, pseudomonas nitritireducen, and Shewanella putrefaciens strain CN32.

2. The porous composite bacterial agent according to claim 1, characterized in that, The modified porous mineral is prepared from the following raw materials: natural porous mineral, crop straw, active organic matter, composite fiber, and water.

3. The porous composite microbial agent according to claim 2, 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 2-5m 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 m 2 / g.

4. The porous composite microbial agent according to claim 2, wherein The natural porous mineral is selected from at least one of perlite, vermiculite, diatomite, volcanic stone, medical stone, and zeolite; And / or, the active organic matter is selected from at least one of xylitol, maltitol, and sorbitol; And / or, 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.

5. The porous composite microbial agent according to any one of claims 2-4, characterized in that, The modified porous mineral is prepared by a method including the following steps: Mix the natural porous mineral and crop straw, and pyrolyze and carbonize to obtain a porous mineral with a biochar layer; Mix the active organic matter, composite fiber, and water, add the porous mineral with a biochar layer, and dry to obtain the modified porous mineral.

6. The porous composite microbial agent according to claim 5, wherein The mass ratio of the natural porous mineral to the crop straw is (5 - 25):1; And / or, the heating program for pyrolysis and carbonization is: heat at 120 - 170 °C for 30 - 60 min, and then raise the temperature to 700 - 850 °C and heat for 30 - 60 s; And / or, the mass ratio of the active organic matter, composite fiber, water, and porous mineral with a biochar layer is 1:(5 - 15): (60-110):(500-850)。 7. The preparation method of the porous composite microbial agent according to any one of claims 1-6, characterized in that, Including the following steps: Co-culture the modified porous mineral with microorganisms having nitrate reduction ability to obtain the porous composite microbial agent.

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

9. Application of the porous composite microbial agent according to any one of claims 1 - 6 in enhancing the process of soil nitrate dissimilatory reduction to ammonium.

10. The application according to claim 9, wherein 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: The 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.

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