CN109797120A - The preparation method and applications of the probiotics of nitrate in a kind of removal soil - Google Patents

The preparation method and applications of the probiotics of nitrate in a kind of removal soil Download PDF

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CN109797120A
CN109797120A CN201910180309.6A CN201910180309A CN109797120A CN 109797120 A CN109797120 A CN 109797120A CN 201910180309 A CN201910180309 A CN 201910180309A CN 109797120 A CN109797120 A CN 109797120A
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nitrate
probiotics
soil
charcoal
denitrifying bacteria
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邵雅璐
湛伟勇
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Wuhan University WHU
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Wuhan University WHU
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Abstract

The invention discloses a kind of preparation method and applications of the probiotics of nitrate in removal soil, belong to technical field of soil remediation.The present invention passes through post-directed training heterotroph denitrifying bacteria, and it is compounded with EM bacterium stoste, fulvic acid, compositional liquor is immobilized on the porous carrier made of the materials such as activation plant ash, charcoal, Nanoscale Iron and kaolin again, the probiotics for removing nitrate in soil are made.Specific preparation step are as follows: (1) domestication and post-directed training of denitrifying bacteria;(2) preparation of denitrifying bacteria-EM compounding agent;(3) preparation of immobilization nano-carrier;(4) preparation of probiotics.Probiotics prepared by the present invention are without side-effects to soil, denitrifying bacteria activity is high, based on heterotrophic organism denitrification and chemical denitrification, nitrate removal significant effect in soil, removal rate reduces nitrate in the intracorporal accumulation of vegetables and risk to underground water leaching up to 85.79%.

Description

The preparation method and applications of the probiotics of nitrate in a kind of removal soil
Technical field
The invention belongs to soil maintenance technical fields, and in particular to a kind of to remove the probiotics of nitrate in soil Preparation method and applications.
Background technique:
Due to a large amount of uses and sewage irrigation of chemical nitrogen fertilizer in facility cultivation, so that Nitrate Accumulation and continuous in soil It enters in underground water, underground water pollution and food-safety problem is caused to become increasingly conspicuous, directly or indirectly affect the mankind's Living environment and health.Facility nitric acid salt form secondary salinization soil can not only bring direct danger with high salt to plant growth Evil, also makes soil beneficial microbe fauna unbalance, even soil can be made to lose production capacity when serious.
The nitrate removed in various water bodys has been applied successfully in biological denitrification method, and nitrogen cycle in nature is utilized in it Denitrification process, being acted on by heterotrophic denitrification by nitrate transformation by denitrifying bacteria is nitrogen.Heterotrophic denitrifying Bacteria is anti- It needs organic carbon source as electron donor in nitrifying process, could be efficiently gas nitrate reduction under suitable carbon-nitrogen ratio Body.It is main raw material(s) that ancestor's virtue etc., which is reported using agricultural crop straw and Zero-valent Iron, is aided with high osmosis material and plasticity bonding Material, is developed a kind of for removing the composite slow release functional material of nitrate in groundwater by different ratio, is researched and developed compound Slow-release function material has certain intensity and porosity, slowly can constantly discharge carbon source.
Nitrate home to return in soil is mainly plant absorption, with Loss in Runoff, leaching to underground water and nitrification and anti-nitre Change effect, it is minimum by side effect of the denitrification removal nitrate to ecological environment for the soil of high Nitrate Accumulation, So soil in-situ microorganism remediation technology is that a kind of have development potentiality, high-efficient and small investment environment remediation technology.It is in situ The product of microorganism remediation technology, process, effect increasingly become the hot spot of concern.Research shows that thin with denitrification from Nanoscale Iron The coupling physical efficiency separated in the compound system of bacterium completely removed nitrate in 5 day time.Schipper is with nano zero-valence Iron powder wall combination electro reclamation renovates the soil by azotate pollution, the nano grade iron powder for the use of mass percent being 0.28% (50~80nm) can achieve 98.06% NO3 -- N degradation rate.Li Tongyan etc. research shows that, locate in advance through 3% sodium hydroxide solution Solid-phase carbon source of the cornstalk of reason as in-situ immobilization all has higher nitrate removal rate, highest under different nitrogen loads Reach 97.17%.
Into soil, there is the EM preparation of denitrification to add although nitrate concentration can be reduced for orientation addition The liquid EM preparation being added in soil, its autologous growth breeding life cycle in can natural death or it is like a fish out of water and Death causes the removal speed to nitrate in soil slower.The NO accumulated in soil3 -- N and NO2 -The substances such as-N as can With microorganism formulation close contact, it is beneficial to the removal of nitrate.Currently, building bioreactor enhancement microbiological effect is repaired The research and application of multiple obstacle soil at home and abroad have more report, such as fix denitrification with bamboo charcoal and activated carbon adsorption method Bacterium removes nitrate etc..Although these researchs achieve certain effect, but still Shortcomings, such as strain used are single, steady Qualitative difference is easy death, and that there are porositys is low for immobilization material, specific surface area is small, adsorption capacity is poor, use cost is high, processing The disadvantages of complex process.
Bioreactor effect and the characteristic of used carrier are closely related, and good carrier can not only greatly improve therein Microorganism concn and quantity, and the activity of microorganism is stronger.Carrier depends on its specific surface to the adsorption capacity of microorganism Product, pore structure and surface characteristic etc..In summary, suitable microorganism and its carrier is selected to utilize bioreactor technology It removes quite important in the nitrate in soil.In consideration of it, the present invention passes through biological domestication and rejuvenation technique, denitrifying bacteria It is immobilized onto the nanosizing carrier of scientific compatibility, the efficiency by promoting heterotrophic denitrification effect obtains the nitrate in soil Efficiently to remove, the pollution caused by nitrate loss and leaching to surrounding water can be effectively avoided, in prevention and control plant Nitrate Accumulation and protection water ecological setting are significant.
Summary of the invention
Goal of the invention: the Nitrate accumulation in soil and its ecology that chemical fertilizer causes are applied due to unreasonable in industrialized agriculture Environment influence, by microorganism orient domestication and compounded technology combined with carrier nanocrystallization technology in the way of construct miniature organism Reactor has developed a kind of probiotics on the basis of experiment, which can effectively remove the nitre in facility obstacle soil Hydrochlorate reduces nitrate in the intracorporal accumulation of vegetables and loss leaching risk.
Technical solution: to realize the above-mentioned technical purpose, the present invention provides a kind of Tiny ecosystems of nitrate in removal soil The preparation method of preparation, includes the following steps:
(1) denitrification flora is inoculated in fermentor, inoculative proportion 8~10%, supplementing culture medium in fermentor, is cultivated Base is mainly made of molasses, fish meal, zinc sulfate, sodium acetate, magnesium sulfate and potassium dihydrogen phosphate, and temperature is controlled at 30~35 DEG C, often Primary, each 10min every 6.5~8h stirring, while sampling Detection micro organism quantity, when denitrification flora bacterial number reaches one EM activity bacterium solution is added when definite value, is cultivated under aerobic fermentation condition, and temperature is controlled at 30~35 DEG C, stirs one every 5~6h It is secondary, each 15min, while sampling Detection micro organism quantity obtains the micro- life of compound denitrification until micro organism quantity reaches certain value Object microbial inoculum.
(2) by compound denitrifying microorganism microbial inoculum, EM bacterium stoste and fulvic acid according to 1~2:0.8~1.2:0.2~0.8 Volume ratio compounding, and be transferred in reaction flask and fermented 3~5 days, denitrifying bacteria-EM compounding agent is made;
(3) plant ash and charcoal are mixed according to a certain volume, is then activated with sodium hydroxide, by the vegetation after activation Ash and charcoal mixture, nanometer iron powder and kaolin are cleaned with deionized water, in mass ratio 60%~70%:5%~10%: 20%~35% mixes, and compresses and granulate, and obtains granular immobilization nano-carrier;
(4) after granular immobilization nano-carrier obtained above being crushed and denitrifying bacteria-EM compounding agent in mass ratio 1~2:8~9 is mixed, and is moved it into centrifugal bottle, and constant incubator is put into, and is vibrated 12-16 hours, is shaken in confined conditions Bed revolving speed is 150-200r/min, is centrifuged on supercentrifuge, and taking-up solid phase components, which are placed in baking oven, dries 1~2 day, is obtained To probiotics.
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, in step (1), denitrification flora It is mainly made of pseudomonas, alcaligenes, Bacillaceae, fiber Acarasiales section, bacteria containing amount is 5.9 × 1010cfu·g-1
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, in step (3), plant ash and life The activation step of object charcoal are as follows: in terms of 100g plant ash and charcoal, be added mass concentration be 12.3% sodium hydroxide 40~ 50mL, carries out the activation of plant ash and charcoal, and activation time is 1~2 day.
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, in step (3), nanometer iron powder Partial size is 50-55nm, and the partial size of granular immobilization nano-carrier is 1~2mm.
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, in the step (3), the life Object charcoal is straw biological charcoal, is prepared by rice straw and corn stover by 1:1 Pintsch process.
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, in the step (2), fulvic acid Concentration be 50gL-1
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, in the step (1), when micro- life Object quantity reaches 109cfu·mL-1When, the stable compound denitrifying microorganism between 4.5~5.0 of pH range can be obtained Microbial inoculum.
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, in the step (1), when anti-nitre Change bacterial number and reaches 106cfu·mL-1When add EM activity bacterium solution, the volume ratio of denitrifying bacteria and EM activity bacterium solution is 1:1。
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil in the step (3), crosses 20 mesh 1:1 is mixed the plant ash and charcoal of sieve by volume.
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil will be above-mentioned in the step (4) Granular immobilization nano-carrier obtained smashes it through 60 meshes, and constant incubator is kept for 35 DEG C, dries 1~2 in 35 DEG C of baking ovens It, obtains probiotics.
The preparation method of the probiotics of nitrate, the EM bacterium stoste mainly contain in a kind of above-mentioned removal soil There are lactic acid bacteria, saccharomycete, photosynthetic bacteria, viable count is no less than 100,000,000/milliliter, and pH is not less than 3.8, and color is yellowish-brown, translucent Liquid, smell are concentrated acid taste or tart flavour.
The preparation method of the probiotics of nitrate in a kind of above-mentioned removal soil, the EM activity bacterium solution is by such as Lower step obtains: 5 parts of EM bacterium stoste, 5 parts of brown sugar, 90 parts of rice washing water are uniformly mixed, room temperature under confined conditions, put Enter rotary shaker 3~5d of anaerobic fermentation that speed is 150~180rpm, stand, is made after being filtered on supercentrifuge.
Probiotics described in any of the above item can be used for removing nitrate in soil and reduce nitrate in vegetables body Interior accumulation and leaching to underground water risk.In use, spreading fertilizer over the fields probiotics when site preparation is transplanted containing high nitric acid In the obstacle soil of salt.In application, preferably, the dosage of probiotics is 3~5kg/667m2
The utility model has the advantages that compared with prior art, the present invention has the advantage that
(1) present invention selects the denitrifying bacteria-EM compounding agent of rejuvenation, it has very strong stability, oxidation resistance Strong and aerobic and anaerobism denitrifying bacteria A wide selection of colours and designs;It wherein joined fulvic acid, greatly strengthen to nitrate in soil Removal effect.It selects nanometer iron powder and activation plant ash and charcoal, kaolin to be mixed with fixation support simultaneously, utilizes High-specific surface area (the 600-800m of nanometer iron powder2·g-1) and activation plant ash and charcoal powerful adsorption capacity so that attached Micro organism quantity and activity greatly increase, carrier of the invention have better biocompatibility and denitrification capability.
(2) preparation method simple process of the invention, production cost is low, have can remove rapidly nitrate in soil, The advantages that without side-effects to soil environment;
(3) denitrifying bacteria-EM compounding agent used in, constructs aerobic and anaerobic bacteria symbiosis environment, and microorganism adapts to soil The ability of earth environment is stronger, makes heterotrophic nitrification and denitrification alternately, and denitrification nitrogen significant effect can reach 85.79% removal rate;
(4) Nanoscale Iron is added in carrier manufacturing process, so that carrier has bigger specific surface area and better biology Compatibility, and zero-valent metal iron has chemical denitrification, the effect of enhancing removal nitrate;
(5) plant ash and charcoal after alkali activates are added in carrier manufacturing process, so that carrier has honeycomb Structure and slow release carbon source, denitrifying bacteria absorption and metabolic capability are promoted, and probiotics are launched to soil to be processed Afterwards, the rapid adapted soil environment of miniature organism reactor energy, efficiently removes nitrate.
Specific embodiment
The various raw materials that the present invention uses are purchased from associated companies and chemical reagent shop, and rice washing water is self-control, below By specific embodiment, the present invention will be described in detail.The denitrifying bacterium is purchased from Guangxi Hong Chun Environmental Protection Technology Co., Ltd.EM Bacterium stoste is purchased from peaceful happy environment friendly biological technology (Nanjing) Co., Ltd of love.
Embodiment 1
(1) denitrifying bacteria is inoculated in fermentor, inoculative proportion 8~10%, supplementing culture medium molasses in fermentor (0.3g·L-1), fish meal (0.5gL-1), zinc sulfate (0.05gL-1), sodium acetate (0.05gL-1), magnesium sulfate (0.02g L-1) and potassium dihydrogen phosphate (0.02gL-1) etc. nutrition, temperature is controlled at 30~35 DEG C, primary every 6.5~8h stirring, every time 10min, while sampling Detection micro organism quantity, when denitrifying bacteria quantity reaches 106cfu·mL-1When add EM active bacteria Liquid (1:1;V:V), to be cultivated under aerobic fermentation condition, temperature is controlled at 30~35 DEG C, and it is primary every 5~6h stirring, every time 15min, while sampling Detection micro organism quantity, when micro organism quantity reaches 109cfu·mL-1When, it is steady that pH range can be obtained The compound denitrifying microorganism microbial inoculum being scheduled between 4.5~5.0.Denitrifying bacteria is by pseudomonas, alcaligenes, bud The denitrification flora of the compositions such as spore Bacteriaceae, fiber Acarasiales section, bacteria containing amount are 5.9 × 1010cfu·g-1, EM bacterium stoste contains cream The effective actives microorganism such as sour bacterium, saccharomycete, photosynthetic bacteria, viable count are no less than 100,000,000/milliliter, in compound denitrifying microorganism Microbial inoculum domestication and rejuvenation during, the effect of nutrient chemical, brown sugar and rice washing water be provided for microbial metabolism the source C, the energy and Inorganic nutrients, aerobic fermentation increase denitrifying bacteria activity and quantity therein.
(2) by compound denitrifying microorganism microbial inoculum, EM bacterium stoste and fulvic acid according to 1~2:0.8~1.2:0.2~0.8 Volume ratio compounding, and be transferred in reaction flask and fermented 3~5 days, denitrifying bacteria-EM compounding agent is made;Fulvic acid is being sent out The meiosis of active material stimulation denitrifying bacteria, the denitrifying bacteria-EM compounding agent through scientific compatibility are generated during ferment The activity and content for further making denitrifying bacteria are improved;
(3) plant ash and charcoal (1:1 of 20 meshes will be crossed;V:V it) mixes, is then activated, will be activated with sodium hydroxide Plant ash and charcoal mixture, nanometer iron powder and kaolin afterwards is cleaned with deionized water, and in mass ratio 60%~70%: 5%~10%:20%~35% is mixed, and is compressed and granulated, and granular immobilization nano-carrier is obtained;Kaolin can be in carrier Good caking property is provided in globulation, so that carrier is not easily broken;
(4) granular immobilization nano-carrier obtained above be crushed into 60 meshes and denitrifying bacteria-EM compounding agent is pressed 1~2:8 of mass ratio~9 is mixed, and is moved it into centrifugal bottle, is put into 35 DEG C of constant incubators and is vibrated in confined conditions 12-16 hours, shaking speed 150-200r/min was centrifuged on supercentrifuge, is taken out solid phase components and is placed on 35 DEG C of bakings It is dried 1~2 day in case, obtains probiotics.
Preferably, in step (3), the plant ash and charcoal (1:1;V:V) mixture crosses 20 meshes, and described receives Rice iron powder partial size is 50~55nm.Plant ash and charcoal provide carbon source and inorganic nutrients, nanometer iron powder tool for microbial metabolism There are huge specific surface area and porous feature, has preferable biocompatibility, and cell large amount of adsorption, microorganism can be effectively improved Adsorb and fix and inhabit, while zero-valent metal have chemical denitrification.
In step (3), the activation step of plant ash and biological carbon mixture are as follows: in terms of 100g plant ash and charcoal, add Enter 40~50mL of sodium hydroxide that mass concentration is 12.3%, carry out the activation of plant ash and charcoal, activation time is 1~2 It.Plant ash and charcoal after activation have honeycomb shape structure, and porosity and specific surface area increase, and have stronger adsorption energy Power is conducive to slow release carbon source and inorganic nutrients and grows for denitrifying bacteria.
In step (3), the diameter of granular fixation support is 1~2mm.However support shapes of the invention are not limited to grain Shape.
Present invention further proposes above-mentioned probiotics to exist for removing nitrate and reduction nitrate in soil Purposes in the intracorporal accumulation of vegetables and leaching risk.
For removing, nitrate and reduction nitrate are in the intracorporal accumulation of vegetables and leaching risk in soil, by micro- life State preparation is spread fertilizer over the fields in the obstacle soil containing high nitrate when site preparation is transplanted.The dosage of probiotics is 3~5kg/ 667m2
Embodiment 2
Denitrifying bacteria is inoculated in fermentor, inoculative proportion 8%, supplementing culture medium molasses (0.3g in fermentor L-1), fish meal (0.5gL-1), zinc sulfate (0.05gL-1), sodium acetate (0.05gL-1), magnesium sulfate (0.02gL-1) and Potassium dihydrogen phosphate (0.02gL-1) etc. nutrition, temperature control at 30 DEG C, primary every 7h stirring, each 10min samples simultaneously Micro organism quantity is detected, when denitrifying bacteria quantity reaches 106cfu·mL-1When add EM activity bacterium solution (1:1;V:V), exist It is cultivated under aerobic fermentation condition, temperature is controlled at 30 DEG C, each 15min primary every 5h stirring, while sampling Detection microorganism Quantity, when micro organism quantity reaches 109cfu·mL-1When to get compound denitrifying microorganism microbial inoculum.By the micro- life of compound denitrification Object microbial inoculum, EM bacterium stoste are compounded with fulvic acid according to the volume ratio of 2.0:1.0:0.6, and are transferred in reaction flask and are fermented 4 days, Denitrifying bacteria-EM compounding agent is made;The plant ash and charcoal (1:1 of 20 meshes will be crossed;V:V it) mixes, then with 12.3% Sodium hydroxide activates 1 day, and plant ash and charcoal mixture, the nanometer iron powder and kaolin deionized water after activation is clear It washes, 65%:5%:30% in mass ratio is mixed, and is compressed and granulated, and granular immobilization nano-carrier, partial size 2mm are obtained.It will Granular immobilization nano-carrier obtained crushed 60 meshes and denitrifying bacteria-EM compounding agent 1:9 in mass ratio mixing, and It moves it into centrifugal bottle, is put into 35 DEG C of constant incubators and vibrates in confined conditions 15 hours, shaking speed 180r/ Min is centrifuged on supercentrifuge, and taking-up solid phase components, which are placed in 35 DEG C of baking ovens, dries 2 days, obtains probiotics.
Carry out pakchoi planting experiment in the facility plastic greenhouse of Nanjing Institute of Vegetable Science's continuous cropping 5 years, if at 4 kinds Reason: (1) it compares;(2) denitrifying bacteria pulvis;(3) EM glass cassie, the production of Nanjing Ai Mule environment friendly biological Technology Co., Ltd.; (4) probiotics of the present invention;The amount of application for handling (2), processing (3) and processing (4) is 4kg/667m2, transplanted in site preparation When spread fertilizer over the fields in soil.3 repetitions of each processing, random alignment.Soil sample is acquired after pakchoi harvest and the monitoring of plant sample is related Index, the results are shown in Table 1.
Table 1 respectively handles the content of soil and the nitrate nitrogen in pakchoi
It is computed and knows, the present embodiment probiotics are 83.08% to the removal rate of soil nitrate N, to plant nitre state N's Removal rate is 93.46%, and the removal rate to underground water nitre state N is 92.38%.As it can be seen from table 1 Tiny ecosystem system of the invention Agent removes nitrate significant effect in soil and is better than EM glass cassie and denitrifying bacteria pulvis, and has preferable buffering to make pH With soluble organic fraction capable of being made to have certain decline, the nitrate nitrogen in plant and underground water also has apparent reduction.
Embodiment 3
Denitrifying bacteria is inoculated in fermentor, inoculative proportion 10%, supplementing culture medium molasses (0.3g in fermentor L-1), fish meal (0.5gL-1), zinc sulfate (0.05gL-1), sodium acetate (0.05gL-1), magnesium sulfate (0.02gL-1) and Potassium dihydrogen phosphate (0.02gL-1) etc. nutrition, temperature control at 35 DEG C, primary every 8h stirring, each 10min samples simultaneously Micro organism quantity is detected, when denitrifying bacteria quantity reaches 106cfu·mL-1Shi Tianjia EM activity bacterium solution (1:1;V:V), good It is cultivated under the conditions of aerobe fermentation, temperature is controlled at 32 DEG C, each 15min primary every 6h stirring, while sampling Detection microbe quantity Amount, when micro organism quantity reaches 109cfu·mL-1When to get compound denitrifying microorganism microbial inoculum.By the micro- life of compound denitrification Object microbial inoculum, EM bacterium stoste are compounded with fulvic acid according to the volume ratio of 1:0.8:0.2, and are transferred in reaction flask and are fermented 3 days, system At denitrifying bacteria-EM compounding agent;The plant ash and charcoal (1:1 of 20 meshes will be crossed;V:V it) mixes, then uses quality percentage Than being activated 2 days for 12.3% sodium hydroxide, plant ash and charcoal mixture, the nanometer iron powder and kaolin after activation are used Deionized water cleaning, 70%:8%:32% is mixed by mass percentage, and is compressed and granulated, and is obtained granular immobilization nanometer and is carried Body, partial size 1mm.Granular immobilization nano-carrier obtained be crushed into 60 meshes and denitrifying bacteria-EM compounding agent by matter Amount is mixed than 2:8, and is moved it into centrifugal bottle, is put into 35 DEG C of constant incubators and is vibrated in confined conditions 12 hours, shakes Bed revolving speed is 160r/min, is centrifuged on supercentrifuge, and taking-up solid phase components, which are placed in 35 DEG C of baking ovens, dries 1 day, is obtained Probiotics.
Carry out pakchoi planting experiment in the facility plastic greenhouse of Nanjing Institute of Vegetable Science's continuous cropping 8 years, if at 4 kinds Reason: (1) it compares;(2) denitrifying bacteria pulvis;(3) EM glass cassie, the production of Nanjing Ai Mule environment friendly biological Technology Co., Ltd.; (4) probiotics of the present invention;The amount of application for handling (2), processing (3) and processing (4) is 5kg/667m2, transplanted in site preparation When spread fertilizer over the fields in soil.3 repetitions of each processing, random alignment.Soil sample is acquired after pakchoi harvest and the monitoring of plant sample is related Index, the results are shown in Table 2.
Table 2 respectively handles the content and yield of soil, underground water and the nitrate nitrogen in pakchoi
It is learnt by the calculating of table 2, the present embodiment probiotics are 85.79% to the removal rate of soil nitrate N, to plant nitre The removal rate of state N is 93.04%, is 92.15% to the removal rate of underground water nitre state N, and the rate of growth of pakchoi is up to 316%.With Upper monitoring data illustrate that nitrate significant effect is better than EM glass cassie and denitrification in probiotics removal soil of the invention Bacterium pulvis has preferable buffer function to compare with control pH, and the nitrate nitrogen in plant and underground water also has apparent drop Low, the yield of pakchoi has obvious rising, significantly alleviates continuous cropping soil nitrate secondary salinization obstacle.
The foregoing is merely exemplary embodiments of the invention, are not intended to limit the invention, it is all in spirit of the invention and Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within principle.

Claims (10)

1. the preparation method of the probiotics of nitrate in a kind of removal soil, which comprises the steps of:
(1) denitrification flora is inoculated in fermentor, inoculative proportion 8~10%, supplementing culture medium in fermentor, culture medium master It to be made of molasses, fish meal, zinc sulfate, sodium acetate, magnesium sulfate and potassium dihydrogen phosphate, temperature is controlled at 30~35 DEG C, every 6.5 ~8h stirring is primary, each 10min, while sampling Detection micro organism quantity, when denitrification flora bacterial number reaches certain value Shi Tianjia EM activity bacterium solution, is cultivated under aerobic fermentation condition, and temperature is controlled at 30~35 DEG C, primary every 5~6h stirring, often Secondary 15min, while sampling Detection micro organism quantity obtain compound denitrifying microorganism bacterium until micro organism quantity reaches certain value Agent;
(2) by compound denitrifying microorganism microbial inoculum, EM bacterium stoste and fulvic acid according to 1~2:0.8~1.2:0.2~0.8 body Product is transferred in reaction flask and is fermented 3~5 days than compounding, and denitrifying bacteria-EM compounding agent is made;
(3) plant ash and charcoal are mixed according to a certain volume, are then activated with sodium hydroxide, by after activation plant ash and Charcoal mixture, nanometer iron powder and kaolin are cleaned with deionized water, 60%~70%:5% in mass ratio~10%:20% ~35% mixes, and compresses and granulate, and obtains granular immobilization nano-carrier;
(4) after granular immobilization nano-carrier obtained above being crushed and denitrifying bacteria-EM compounding agent in mass ratio 1~ 2:8~9 is mixed, and is moved it into centrifugal bottle, and constant incubator is put into, and is vibrated 12-16 hours in confined conditions, and shaking table turns Speed is 150-200r/min, is centrifuged on supercentrifuge, and taking-up solid phase components, which are placed in baking oven, dries 1~2 day, is obtained micro- Ecological agent.
2. the preparation method of the probiotics of nitrate in a kind of removal soil as described in claim 1, which is characterized in that In step (1), denitrification flora is mainly made of pseudomonas, alcaligenes, Bacillaceae, fiber Acarasiales section, is contained Bacterium amount is 5.9 × 1010cfu·g-1
3. the preparation method of the probiotics of nitrate in a kind of removal soil as described in claim 1, which is characterized in that In step (3), the activation step of plant ash and charcoal are as follows: in terms of 100g plant ash and charcoal, mass concentration, which is added, is 12.3% 40~50mL of sodium hydroxide, carries out the activation of plant ash and charcoal, and activation time is 1~2 day.
4. the preparation method of the probiotics of nitrate in a kind of removal soil as described in claim 1, which is characterized in that In step (3), the partial size of nanometer iron powder is 50-55nm, and the partial size of granular immobilization nano-carrier is 1~2mm.
5. the preparation method of the probiotics of nitrate in a kind of removal soil as described in claim 1, which is characterized in that In the step (3), the charcoal is straw biological charcoal, prepared by rice straw and corn stover by 1:1 Pintsch process and ?.
6. the preparation method of the probiotics of nitrate in a kind of removal soil as described in claim 1, which is characterized in that In the step (2), the concentration of fulvic acid is 50gL-1
7. the preparation method of the probiotics of nitrate in a kind of removal soil as described in claim 1, which is characterized in that In the step (1), when micro organism quantity reaches 109cfu·mL-1When, can be obtained pH range stablize 4.5~5.0 it Between compound denitrifying microorganism microbial inoculum.
8. the described in any item probiotics of claim 1~7 can be used for removing nitrate and reduction nitrate in soil and exist The risk of vegetables intracorporal accumulation and leaching to underground water.
9. application as claimed in claim 8, which is characterized in that in use, probiotics are spread fertilizer over the fields when site preparation is transplanted Into the obstacle soil containing high nitrate.
10. application as claimed in claim 9, which is characterized in that the dosage of probiotics is 3~5kg/667m2
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