CN109797120B - Preparation method and application of microecological preparation for removing nitrate in soil - Google Patents
Preparation method and application of microecological preparation for removing nitrate in soil Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a microecological preparation for removing nitrate in soil, and belongs to the technical field of soil remediation. The invention prepares the microecological preparation for removing nitrate in soil by directionally culturing heterotrophic denitrifying bacteria, compounding with EM (effective microorganisms) bacteria stock solution and fulvic acid, and immobilizing compound solution on a porous carrier made of materials such as activated plant ash, biochar, nano iron, kaolin and the like. The preparation method comprises the following specific steps: (1) domestication and directional culture of denitrifying bacteria; (2) preparing a denitrifying bacterium-EM compound agent; (3) preparing an immobilized nano carrier; (4) and (4) preparing a microecological preparation. The microecological preparation prepared by the invention has no side effect on soil, has high denitrifying bacterial activity, has obvious effect of removing nitrate in soil based on the denitrification of heterotrophic organisms and chemical denitrification, has the removal rate of 85.79 percent, and reduces the risk of accumulation of nitrate in vegetables and leaching of nitrate into underground water.
Description
Technical Field
The invention belongs to the technical field of soil remediation, and particularly relates to a preparation method and application of a microecological preparation for removing nitrate in soil.
Background art:
in the facility cultivation, due to the use of a large amount of chemical nitrogen fertilizers and sewage irrigation, nitrate in soil is accumulated and continuously enters underground water, so that the problems of underground water pollution and food safety are increasingly highlighted, and the living environment and the body health of human beings are directly or indirectly influenced. The facility nitrate type secondary salinized soil not only brings direct high-salt harm to the growth of crops, but also unbalance beneficial microbial flora of the soil, and even can lose the production capacity of the soil in serious cases.
The biological denitrification method has been successfully applied to remove the nitrate in various water bodies, and utilizes the denitrification process of nitrogen circulation in the nature to convert the nitrate into nitrogen by denitrifying bacteria through heterotrophic denitrification. Heterotrophic denitrifying bacteria need an organic carbon source as an electron donor in the denitrification process, and can efficiently reduce nitrate into gas under a proper carbon-nitrogen ratio. The reports of the Zongfang and the like use crop straws and zero-valent iron as main raw materials, and high-permeability materials and plastic bonding materials are used as auxiliary materials, a composite slow-release functional material for removing nitrate in underground water is developed according to different proportions, and the developed composite slow-release functional material has certain strength and porosity and can slowly and continuously release a carbon source.
The nitrate in the soil mainly enters the underground water through plant absorption, runoff loss, leaching and nitrification and denitrification, and the nitrate-removing side effect on the ecological environment is minimal for the soil with high nitrate accumulation through denitrification, so the soil in-situ microbial remediation technology is an environment remediation technology with development potential, high efficiency and low investment. Products, processes and effects of in situ microbial remediation technology are becoming more and more a focus of attention. The study showed that the coupling isolated from the complex system of nano-iron and denitrifying bacteria was able to completely remove nitrate in 5 days. The Schipper treats the nitrate-polluted soil by combining a nano zero-valent iron powder wall and an electric restoration method, and uses 0.28 mass percent of nano-scale iron powder (50-80 nm) to achieve 98.06 mass percent of NO3 --N degradation rate. Researches of Limonissian and the like show that the cornstalk pretreated by 3% sodium hydroxide solution has higher nitrate removal rate under different nitrogen loads as an in-situ repaired solid-phase carbon source, and the maximum nitrate removal rate reaches 97.17%.
The directional addition of the EM preparation with denitrification effect to the soil can reduce the concentration of the nitrate, but the liquid EM preparation added to the soil naturally dies or dies without adapting to the environment in the life cycle of self-growth and propagation, so that the removal speed of the nitrate in the soil is slow. NO accumulated in soil3 --N and NO2 -The substances such as-N and the like can be in close contact with the microbial preparation, and nitrate removal is facilitated. At present, research on constructing bioreactor to strengthen microbial action and repair obstacle soilAnd the application has been reported at home and abroad, for example, the denitrifying bacteria are fixed by using bamboo charcoal and activated carbon adsorption methods to remove nitrate. Although the researches have achieved certain effects, the defects still exist, such as single strain, poor stability and easy death, and the immobilized material has the defects of low porosity, small specific surface area, poor adsorption capacity, high use cost, complex processing technology and the like.
The bioreactor effect is closely related to the characteristics of the carrier, and the good carrier can not only greatly improve the concentration and the quantity of microorganisms in the carrier, but also has stronger activity of the microorganisms. The adsorption capacity of the carrier for microorganisms depends on its specific surface area, pore structure, surface characteristics, and the like. In summary, it is important to select suitable microorganisms and carriers for them in the removal of nitrate from soil using bioreactor technology. In view of the above, the invention immobilizes denitrifying bacteria in a scientifically compatible nano-carrier by means of biological domestication and rejuvenation technology, efficiently removes nitrate in soil by improving the efficiency of heterotrophic denitrification, can effectively avoid pollution to surrounding water bodies caused by nitrate loss and leaching, and has great significance for preventing and controlling nitrate accumulation in plants and protecting the ecological environment of the water bodies.
Disclosure of Invention
The purpose of the invention is as follows: aiming at soil nitrate accumulation and ecological environment influence caused by unreasonable fertilizer application in facility agriculture, a micro bioreactor is constructed by combining a microorganism directional domestication and compounding technology and a carrier nanocrystallization technology, and a microecological preparation is developed on the basis of an experiment.
The technical scheme is as follows: in order to achieve the technical purpose, the invention provides a preparation method of a microecological preparation for removing nitrate in soil, which comprises the following steps:
(1) inoculating denitrifying bacteria groups into a fermentation tank, wherein the inoculation proportion is 8-10%, supplementing a culture medium in the fermentation tank, wherein the culture medium mainly comprises molasses, fish meal, zinc sulfate, sodium acetate, magnesium sulfate and potassium dihydrogen phosphate, the temperature is controlled to be 30-35 ℃, the fermentation tank is stirred once every 6.5-8 hours, the number of microorganisms is sampled and detected every 10 minutes, EM active bacteria liquid is added when the number of denitrifying bacteria groups reaches a certain value, the fermentation tank is cultured under the aerobic fermentation condition, the temperature is controlled to be 30-35 ℃, the fermentation tank is stirred once every 5-6 hours, the number of microorganisms is sampled and detected every 15 minutes, and the composite denitrifying microorganism agent is obtained until the number of microorganisms reaches a certain value.
(2) Compounding the composite denitrifying microbial agent, the EM bacteria stock solution and the fulvic acid according to the volume ratio of 1-2: 0.8-1.2: 0.2-0.8, and transferring the mixture into a reaction bottle for fermentation for 3-5 days to prepare a denitrifying bacteria-EM compound agent;
(3) uniformly mixing plant ash and biochar according to a certain volume ratio, activating with sodium hydroxide, and cleaning the mixture of the activated plant ash and biochar, nano iron powder and kaolin with deionized water according to a mass ratio of 60-70%: 5% -10%: 20 to 35 percent of the mixture is mixed evenly and compressed into particles to obtain the granular immobilized nano-carrier;
(4) crushing the prepared granular immobilized nano-carrier, mixing the crushed granular immobilized nano-carrier with a denitrifying bacteria-EM compound agent according to a mass ratio of 1-2: 8-9, transferring the mixture into a centrifugal bottle, putting the centrifugal bottle into a constant-temperature incubator, oscillating the flask under a sealed condition for 12-16 hours at a shaking table rotation speed of 150-.
In the step (1), the denitrifying bacteria group mainly comprises pseudomonas, alcaligenes, bacillus and fiber slime bacteria, and the bacteria content is 5.9 x 1010cfu·g-1。
In the above preparation method of the microecological preparation for removing nitrate from soil, in the step (3), the activation steps of the plant ash and the biochar are as follows: adding 40-50 mL of sodium hydroxide with the mass concentration of 12.3% into 100g of plant ash and biochar to activate the plant ash and the biochar for 1-2 days.
In the preparation method of the microecological preparation for removing the nitrate in the soil, in the step (3), the particle size of the nano iron powder is 50-55nm, and the particle size of the granular immobilized nano carrier is 1-2 mm.
In the step (3), the biochar is straw biochar which is prepared from straw stalks and corn stalks according to a ratio of 1:1 high-temperature cracking.
In the above method for preparing a microecological preparation for removing nitrate from soil, in the step (2), the concentration of fulvic acid is 50 g.L-1。
In the above preparation method of the microecological preparation for removing nitrate from soil, in the step (1), when the number of the microorganisms reaches 109cfu·mL-1And then the composite denitrifying microorganism bacterial agent with the pH range stabilized between 4.5 and 5.0 can be obtained.
In the above preparation method of the microecological preparation for removing nitrate from soil, in the step (1), when the number of denitrifying bacteria reaches 106cfu·mL-1And adding EM active bacterial liquid, wherein the volume ratio of the denitrifying bacteria to the EM active bacterial liquid is 1: 1.
In the step (3), the plant ash and the biochar which are sieved by the 20-mesh sieve are uniformly mixed according to the volume ratio of 1: 1.
In the step (4), the prepared granular immobilized nano-carrier is crushed and then sieved by a 60-mesh sieve, and the granular immobilized nano-carrier is dried in a drying oven at 35 ℃ and a constant temperature incubator at 35 ℃ for 1-2 days to obtain the microecological preparation.
The EM bacteria stock solution mainly contains lactic acid bacteria, saccharomycetes and photosynthetic bacteria, the number of viable bacteria is not less than 1 hundred million/ml, the pH value is not less than 3.8, the EM bacteria stock solution is yellow brown, semitransparent liquid and has strong sour or sour smell.
According to the preparation method of the microecological preparation for removing the nitrate in the soil, the EM active bacterial liquid is obtained through the following steps: uniformly mixing 5 parts of EM (effective microorganism) stock solution, 5 parts of brown sugar and 90 parts of rice washing water, putting into a rotary shaking table with the speed of 150-180 rpm under the condition of room temperature sealing for anaerobic fermentation for 3-5 days, standing, and filtering on a high-speed centrifuge to obtain the product.
The microecological agent of any one of the above claims can be used for removing nitrate from soil and reducing the risk of nitrate accumulation in vegetables and leaching to groundwater. In application, the microecologics are scattered into high-nitrate-content obstacle soil during soil preparation and transplantation. When the microbial ecological agent is applied, the dosage of the microbial ecological agent is preferably 3-5 kg/667m2。
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the invention selects the rejuvenation denitrifying bacteria-EM mixture which has strong stability, strong oxidation resistance and complete types of aerobic and anaerobic denitrifying bacteria; the fulvic acid is added, so that the removal effect of nitrate in soil is greatly enhanced. Simultaneously, nano iron powder and activated plant ash are selected to be mixed with the biochar and the kaolin to prepare the immobilized carrier, and the high specific surface area (600-800 m) of the nano iron powder is utilized2·g-1) And the strong adsorption capacity of the activated plant ash and the biochar greatly increases the number and the activity of attached microorganisms, and the carrier has better biocompatibility and denitrification performance.
(2) The preparation method has the advantages of simple process, low production cost, capability of quickly removing nitrate in soil, no side effect on soil environment and the like;
(3) the denitrifying bacteria-EM compound agent constructs an aerobic and anaerobic bacteria symbiotic environment, the capability of microorganisms adapting to the soil environment is stronger, the heterotrophic nitrification and the denitrification are alternately carried out, the effect of removing nitrate nitrogen is obvious, and the removal rate can reach 85.79%;
(4) the nano iron is added in the carrier manufacturing process, so that the carrier has larger specific surface area and better biocompatibility, and the zero-valent metallic iron has chemical denitrification effect, thereby enhancing the effect of removing nitrate;
(5) the plant ash and the biochar which are activated by alkali are added in the manufacturing process of the carrier, so that the carrier has a honeycomb structure and a slow-release carbon source, the adsorption and metabolic capacity of denitrifying bacteria is improved, and after the microbial ecological agent is put into the soil to be treated, the micro bioreactor can quickly adapt to the soil environment and efficiently remove nitrate.
Detailed Description
The raw materials used in the present invention are purchased from related companies and chemical agent stores, and the rice washing water is self-made, and the present invention will be described in detail below by specific examples. The denitrifying bacteria are purchased from environmental protection science and technology limited of Kwangtze Hongkun, Guangxi. EM bacteria stock solution is purchased from environment-friendly biological technology (Nanjing) Co.
Example 1
(1) Inoculating denitrifying bacteria into a fermentation tank, wherein the inoculation proportion is 8-10%, and the fermentation tank is supplemented with culture medium molasses (0.3 g.L)-1) 0.5 g.L fish meal-1) Zinc sulfate (0.05 g. L)-1) Sodium acetate (0.05 g. L)-1) Magnesium sulfate (0.02 g. L)-1) And potassium dihydrogen phosphate (0.02 g. L)-1) When the nutrition is equal, the temperature is controlled to be 30-35 ℃, the mixture is stirred once every 6.5-8 hours, each time is 10min, and the microbial quantity is simultaneously sampled and detected, when the quantity of denitrifying bacteria reaches 106cfu·mL-1Adding EM active bacteria liquid (1: 1; V: V), culturing under aerobic fermentation condition, controlling temperature at 30-35 deg.C, stirring once every 5-6 hr for 15min, simultaneously sampling and detecting microorganism amount, when microorganism amount reaches 109cfu·mL-1And then the composite denitrifying microorganism bacterial agent with the pH range stabilized between 4.5 and 5.0 can be obtained. The denitrifying bacteria is denitrifying bacteria group composed of Pseudomonas, Alcaligenes, Bacillaceae, and Mycosphaerellaceae, and has bacteria content of 5.9 × 1010cfu·g-1The EM bacteria stock solution contains effective active microorganisms such as lactic acid bacteria, saccharomycetes, photosynthetic bacteria and the like, the number of the active bacteria is not less than 1 hundred million/ml, in the domestication and rejuvenation processes of the composite denitrifying microorganism bacteria agent, the culture agent, the brown sugar and the rice washing water have the functions of providing C source, energy and inorganic nutrition for microorganism metabolism, and the activity and the number of the denitrifying bacteria in the culture agent are increased through aerobic fermentation.
(2) Compounding the composite denitrifying microbial agent, the EM bacteria stock solution and the fulvic acid according to the volume ratio of 1-2: 0.8-1.2: 0.2-0.8, and transferring the mixture into a reaction bottle for fermentation for 3-5 days to prepare a denitrifying bacteria-EM compound agent; fulvic acid generates active substances to stimulate meiosis of denitrifying bacteria in the fermentation process, and the activity and content of the denitrifying bacteria are further improved by the scientifically-matched denitrifying bacteria-EM compound agent;
(3) mixing the plant ash and the biochar (1: 1; V: V) which are sieved by a 20-mesh sieve uniformly, activating by using sodium hydroxide, and cleaning the mixture of the activated plant ash and the biochar, the nano iron powder and the kaolin by using deionized water according to the mass ratio of 60-70%: 5% -10%: 20 to 35 percent of the mixture is mixed evenly and compressed into particles to obtain the granular immobilized nano-carrier; the kaolin can provide good cohesiveness in the carrier balling process, so that the carrier is not easy to break;
(4) and (2) crushing the prepared granular immobilized nano-carrier, sieving the crushed granular immobilized nano-carrier by a sieve of 60 meshes, mixing the granular immobilized nano-carrier with a denitrifying bacteria-EM compound agent according to the mass ratio of 1-2: 8-9, transferring the mixture into a centrifugal bottle, putting the centrifugal bottle into a constant-temperature incubator at 35 ℃ and oscillating the mixture for 12-16 hours under a sealed condition, wherein the rotating speed of a shaking table is 200r/min, centrifuging the mixture on a high-speed centrifuge, taking out solid phase components, and then putting the solid phase components into an oven at 35 ℃ and drying the solid phase components for 1-2 days to.
Preferably, in the step (3), the mixture of the plant ash and the biochar (1: 1; V: V) is sieved by a 20-mesh sieve, and the particle size of the nano iron powder is 50-55 nm. The plant ash and the biochar provide carbon sources and inorganic nutrition for microbial metabolism, the nano iron powder has the characteristics of huge specific surface area and porosity, better biocompatibility and large cell adsorption capacity, can effectively improve the adsorption, fixation and inhabitation of microorganisms, and meanwhile, the zero-valent metal has the chemical denitrification effect.
In the step (3), the activation step of the mixture of plant ash and biochar is as follows: adding 40-50 mL of sodium hydroxide with the mass concentration of 12.3% into 100g of plant ash and biochar to activate the plant ash and the biochar for 1-2 days. The activated plant ash and the activated charcoal have honeycomb-shaped structures, the porosity and the specific surface area are increased, the adsorption capacity is stronger, and the slow release of a carbon source and inorganic nutrition is facilitated for the growth of denitrifying bacteria.
In the step (3), the diameter of the granular immobilization carrier is 1 to 2 mm. However, the shape of the carrier of the present invention is not limited to the granular shape.
The invention further provides the application of the microecological preparation in removing nitrate in soil and reducing the accumulation and leaching risk of nitrate in vegetables.
When the micro-ecological preparation is used for removing nitrate in soil and reducing the accumulation and leaching risk of nitrate in vegetables, the micro-ecological preparation is scattered into high-nitrate-content obstacle soil during soil preparation and transplantation. The dosage of the microecological preparation is 3-5 kg/667m2。
Example 2
Denitrifying bacteria were inoculated into a fermenter at an inoculation rate of 8% and supplemented with medium molasses (0.3 g. L)-1) 0.5 g.L fish meal-1) Zinc sulfate (0.05 g. L)-1) Sodium acetate (0.05 g. L)-1) Magnesium sulfate (0.02 g. L)-1) And potassium dihydrogen phosphate (0.02 g. L)-1) When the nutrition is equal, the temperature is controlled at 30 ℃, the mixture is stirred once every 7 hours for 10min every time, and the microbial quantity is sampled and detected at the same time, when the quantity of denitrifying bacteria reaches 106cfu·mL-1Adding EM active bacteria liquid (1: 1; V: V), culturing under aerobic fermentation condition, controlling temperature at 30 deg.C, stirring every 5 hr for 15min, sampling and detecting microorganism amount, when microorganism amount reaches 109cfu·mL-1Then the composite denitrifying microorganism bacterial agent is obtained. Compounding the composite denitrifying microbial agent, the EM bacteria stock solution and the fulvic acid according to the volume ratio of 2.0:1.0:0.6, and fermenting in a reaction bottle for 4 days to prepare a denitrifying bacteria-EM compound agent; mixing the plant ash and the biochar (1: 1; V: V) which are sieved by a 20-mesh sieve uniformly, activating the mixture for 1 day by using 12.3% of sodium hydroxide, and cleaning the mixture of the activated plant ash and the biochar, nano iron powder and kaolin by using deionized water according to a mass ratio of 65%: 5%: mixing 30% of the above components, and compressing into granules with particle size of 2 mm. Pulverizing the obtained granular immobilized nano-carrier, sieving with 60 mesh sieve, and denitrifyingMixing the bacteria-EM mixture according to the mass ratio of 1:9, transferring the mixture into a centrifugal bottle, placing the centrifugal bottle into a constant-temperature incubator at 35 ℃ and oscillating the flask for 15 hours under a closed condition, wherein the rotating speed of a shaking table is 180r/min, centrifuging the mixture on a high-speed centrifuge, taking out solid phase components, and then placing the solid phase components in an oven at 35 ℃ and drying the solid phase components for 2 days to obtain the microecological preparation.
A cabbage planting test is carried out in a facility greenhouse of continuous cropping 5 years at vegetable science research institute of Nanjing, and 4 treatments are set: (1) comparison; (2) denitrifying bacteria powder; (3) EM Becasi, Nanjing Aijigle environmental protection biotechnology Limited; (4) the invention relates to a micro-ecological preparation; the application rates of the treatment (2), the treatment (3) and the treatment (4) are 4kg/667m2And the fertilizer is scattered into soil during soil preparation and transplanting. Each treatment was repeated 3 times and randomly arranged. The soil sample and the plant sample are collected after harvesting the pakchoi to monitor related indexes, and the results are shown in table 1.
TABLE 1 nitrate nitrogen content in the soil and pakchoi treated in each treatment
According to calculation, the removal rate of the microecological preparation in the embodiment to the soil nitrate state N is 83.08%, the removal rate to the plant nitrate state N is 93.46%, and the removal rate to the underground water nitrate state N is 92.38%. As can be seen from Table 1, the microecological preparation of the invention has a significantly better effect of removing nitrate in soil than EM bocasi and denitrifying bacteria powder, has a better buffering effect on pH, can reduce soluble salt to a certain extent, and can obviously reduce nitrate nitrogen in plants and groundwater.
Example 3
Denitrifying bacteria were inoculated into a fermenter at an inoculation rate of 10% and supplemented with medium molasses (0.3 g. L)-1) 0.5 g.L fish meal-1) Zinc sulfate (0.05 g. L)-1) Sodium acetate (0.05 g. L)-1) Magnesium sulfate (0.02 g. L)-1) And potassium dihydrogen phosphate (0.02 g. L)-1) When the nutrition is equal, the temperature is controlled at 35 ℃, the mixture is stirred once every 8 hours for 10min each time, and the microbial quantity is sampled and detected when the denitrifying bacteria countIn an amount of up to 106cfu·mL-1Adding EM active bacteria liquid (1: 1; V: V), culturing under aerobic fermentation condition, controlling temperature at 32 deg.C, stirring once every 6 hr for 15min, simultaneously sampling and detecting microorganism amount, when microorganism amount reaches 109cfu·mL-1And then the composite denitrifying microorganism bacterial agent is obtained. Compounding the composite denitrifying microbial agent, the EM bacteria stock solution and the fulvic acid according to the volume ratio of 1:0.8:0.2, and fermenting in a reaction bottle for 3 days to prepare a denitrifying bacteria-EM compound agent; mixing the plant ash and the biochar (1: 1; V: V) which are sieved by a 20-mesh sieve uniformly, activating the mixture for 2 days by using sodium hydroxide with the mass percent of 12.3%, and cleaning the mixture of the activated plant ash and the biochar, the nano iron powder and the kaolin by using deionized water, wherein the mass percent of the mixture is as follows: 8%: 32 percent of the mixture is mixed evenly and compressed into particles to obtain the granular immobilized nano-carrier with the particle size of 1 mm. Crushing the prepared granular immobilized nano-carrier, sieving the crushed granular immobilized nano-carrier with a 60-mesh sieve, mixing the granular immobilized nano-carrier with a denitrifying bacterium-EM mixture according to the mass ratio of 2:8, transferring the mixture into a centrifugal bottle, putting the centrifugal bottle into a 35 ℃ constant temperature incubator, oscillating the centrifugal bottle under a closed condition for 12 hours at the rotating speed of a shaking table of 160r/min, centrifuging the mixture on a high-speed centrifuge, taking out solid phase components, and then putting the solid phase components into a 35 ℃ oven to dry the solid phase components for 1 day to obtain the microecolog.
A cabbage planting test is developed in a facility greenhouse of 8 years of continuous cropping of vegetable science research institute in Nanjing, and 4 treatments are set: (1) comparison; (2) denitrifying bacteria powder; (3) EM Becasi, Nanjing Aijigle environmental protection biotechnology Limited; (4) the invention relates to a micro-ecological preparation; the application rates of the treatment (2), the treatment (3) and the treatment (4) were 5kg/667m2And the fertilizer is scattered into soil during soil preparation and transplanting. Each treatment was repeated 3 times and randomly arranged. The soil sample and the plant sample are collected after harvesting the pakchoi to monitor the related indexes, and the results are shown in table 2.
TABLE 2 nitrate nitrogen content and yield in the soil, groundwater and pakchoi treated in each treatment
From the calculation of table 2, the microecological preparation of the present embodiment has a removal rate of 85.79% for the soil nitrate N, 93.04% for the plant nitrate N, 92.15% for the groundwater nitrate N, and an increased yield of 316% for pakchoi. The monitoring data show that the effect of the microecological preparation for removing nitrate in soil is obviously superior to that of EM bocasi and denitrifying bacteria powder, the microecological preparation has a better buffering effect on pH, compared with a control, nitrate nitrogen in plants and underground water is also obviously reduced, the yield of pakchoi is obviously increased, and the secondary salinization obstacle of continuous cropping soil nitrate is obviously relieved.
The above description is only exemplary of the present invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A preparation method of a microecological preparation for removing nitrate in soil is characterized by comprising the following steps:
(1) inoculating denitrifying bacteria groups into a fermentation tank, wherein the inoculation proportion is 8-10%, supplementing a culture medium in the fermentation tank, wherein the culture medium consists of molasses, fish meal, zinc sulfate, sodium acetate, magnesium sulfate and potassium dihydrogen phosphate, the temperature is controlled to be 30-35 ℃, the fermentation tank is stirred once every 6.5-8 hours and 10min each time, simultaneously sampling and detecting the microbial count, when the bacterial count of the denitrifying bacteria groups reaches a certain value, adding EM active bacterial liquid, culturing under aerobic fermentation conditions, the temperature is controlled to be 30-35 ℃, the fermentation tank is stirred once every 5-6 hours and 15min each time, simultaneously sampling and detecting the microbial count until the microbial count reaches a certain value, and obtaining the composite denitrifying microbial agent;
(2) compounding the composite denitrifying microbial agent, the EM bacteria stock solution and the fulvic acid according to the volume ratio of 1-2: 0.8-1.2: 0.2-0.8, and transferring the mixture into a reaction bottle for fermentation for 3-5 days to prepare a denitrifying bacteria-EM compound agent;
(3) uniformly mixing plant ash and biochar according to a certain volume ratio, activating with sodium hydroxide, and cleaning the mixture of the activated plant ash and biochar, nano iron powder and kaolin with deionized water, wherein the mass ratio is 60-70%: 5% -10%: 20% -35% of the mixture is uniformly mixed and compressed into particles to obtain the granular immobilized nano-carrier;
(4) crushing the prepared granular immobilized nano-carrier, mixing the crushed granular immobilized nano-carrier with a denitrifying bacteria-EM compound agent according to a mass ratio of 1-2: 8-9, transferring the mixture into a centrifugal bottle, putting the centrifugal bottle into a constant-temperature incubator, oscillating the centrifugal bottle under a closed condition for 12-16 hours at a shaking table rotating speed of 150-;
the EM bacteria stock solution contains lactic acid bacteria, saccharomycetes and photosynthetic bacteria, the number of viable bacteria is not less than 1 hundred million/ml, the pH value is not less than 3.8, the EM bacteria stock solution is yellow brown, is semitransparent liquid and has sour taste.
2. The method for preparing a microecological preparation for removing nitrate from soil according to claim 1, wherein in the step (1), the denitrifying bacteria comprise Pseudomonas, Alcaligenes, Bacillaceae, Mycosphaerellaceae, and have a bacterial content of 5.9 x 1010cfu·g-1。
3. The method for preparing a microecological preparation for removing nitrate from soil according to claim 1, wherein the activation of plant ash and biochar in step (3) comprises: adding 40-50 mL of sodium hydroxide with the mass concentration of 12.3% into 100g of plant ash and biochar to activate the plant ash and the biochar for 1-2 days.
4. The method for preparing a microecological preparation for removing nitrate from soil according to claim 1, wherein in the step (3), the particle size of the nano iron powder is 50-55nm, and the particle size of the granular immobilized nano carrier is 1-2 mm.
5. The method for preparing a microecological preparation for removing nitrate from soil according to claim 1, wherein in the step (3), the biochar is straw biochar comprising rice straw and corn stover in a ratio of 1:1 high-temperature cracking.
6. The method for preparing a microecological preparation for removing nitrate from soil according to claim 1, wherein the concentration of fulvic acid in step (2) is 50 g-L-1。
7. The method for preparing a microecological preparation for removing nitrate from soil according to claim 1, wherein the microbial population in the step (1) reaches 109 cfu· mL-1And then the composite denitrifying microorganism bacterial agent with the pH range stabilized between 4.5 and 5.0 can be obtained.
8. A microecological preparation for removing nitrate from soil is prepared by the preparation method of the microecological preparation for removing nitrate from soil, which is characterized by comprising denitrifying bacteria, EM (effective microorganisms) and immobilized nanocarriers, according to any one of claims 1 to 7;
the immobilized nano-carrier comprises plant ash, charcoal, nano iron powder and kaolin;
the EM bacteria stock solution contains lactic acid bacteria, saccharomycetes and photosynthetic bacteria, the number of viable bacteria is not less than 1 hundred million/ml, the pH value is not less than 3.8, the EM bacteria stock solution is yellow brown, is semitransparent liquid and has sour taste.
9. The use of the microecological agent for the removal of nitrate from soil according to claim 8 for the production increase of pakchoi.
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| CN105347513A (en) * | 2015-12-01 | 2016-02-24 | 南通河海大学海洋与近海工程研究院 | Preparation method and application of microecological preparation |
| CN107512977A (en) * | 2017-09-07 | 2017-12-26 | 河海大学 | A kind of preparation method and applications of EM nano biologicals charcoal base soil conditioner |
| CN108707562A (en) * | 2018-05-24 | 2018-10-26 | 江苏世邦生物工程科技有限公司 | Microorganism formulation and its preparation method and application for repairing three polluted by nitrogen soil |
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| WO2012096462A2 (en) * | 2011-01-14 | 2012-07-19 | 한국해양대학교 산학협력단 | Biological treatment method and waste-water treatment agent for refractory wastewater |
| CN103803703A (en) * | 2014-02-25 | 2014-05-21 | 合肥工业大学 | Method for simultaneously removing phosphorous and nitrogen through synergistic effect of nanoscale-iron and microbes |
| CN105347513A (en) * | 2015-12-01 | 2016-02-24 | 南通河海大学海洋与近海工程研究院 | Preparation method and application of microecological preparation |
| CN107512977A (en) * | 2017-09-07 | 2017-12-26 | 河海大学 | A kind of preparation method and applications of EM nano biologicals charcoal base soil conditioner |
| CN108707562A (en) * | 2018-05-24 | 2018-10-26 | 江苏世邦生物工程科技有限公司 | Microorganism formulation and its preparation method and application for repairing three polluted by nitrogen soil |
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