CN113046087A - Medicament and method for promoting microorganisms to restore soil secondary salinization - Google Patents

Medicament and method for promoting microorganisms to restore soil secondary salinization Download PDF

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CN113046087A
CN113046087A CN202110290156.8A CN202110290156A CN113046087A CN 113046087 A CN113046087 A CN 113046087A CN 202110290156 A CN202110290156 A CN 202110290156A CN 113046087 A CN113046087 A CN 113046087A
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soil
denitrifying bacteria
polysaccharide
algal
promoting
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CN113046087B (en
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李鹏程
张辉
陈晓琳
刘松
于华华
邢荣娥
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K17/00—Soil-conditioning materials or soil-stabilising materials
    • C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
    • C09K17/18—Prepolymers; Macromolecular compounds
    • C09K17/32—Prepolymers; Macromolecular compounds of natural origin, e.g. cellulosic materials
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound

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  • Chemical & Material Sciences (AREA)
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  • Agronomy & Crop Science (AREA)
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Abstract

The invention discloses a medicament and a method for promoting microorganisms to restore secondary salinization of soil. The addition of algal polysaccharide promotes the rapid growth of denitrifying bacteria, can pertinently reduce the nitrate content in soil, simultaneously enhances the capability of the denitrifying bacteria to consume nitrate ions, reduces the accumulation of nitrite, enhances the capability of the denitrifying bacteria to carry out the dissimilatory reduction, reduces the transformation of the nitrate ions to nitrogen, is favorable for balancing the balance of ammonia and nitrate nitrogen, promotes the growth of plants, improves the stress resistance of the plants, simultaneously can provide rich organic matters for the soil, improves the soil structure, increases the water and air permeability of the soil, and is a green, safe and rapid method for relieving the problem of secondary salinization of the soil.

Description

Medicament and method for promoting microorganisms to restore soil secondary salinization
The technical field is as follows:
the invention belongs to the technical field of soil improvement, and relates to a medicament and a method for promoting microorganisms to restore soil secondary salinization.
Background art:
excessive nitrogen fertilizer used in the greenhouse cannot be completely utilized by plants, a large amount of fertilizer is accumulated in soil, so that the salt content in the soil is gradually increased, the nitrate content accounts for a large proportion, the accumulation of a large amount of nitrate ions is the main reason of secondary salinization, and the reduction of the content of the nitrate ions is an effective way for repairing the secondary salinization of the soil.
Denitrification refers to a process of gradually reducing nitrate nitrogen into nitrogen by microorganisms, and the process converts the nitrate nitrogen into gas and discharges the gas into the air, so that the nitrate nitrogen content is effectively reduced. The use of denitrifying microorganisms for denitrification of soil is a very novel idea, but the selection of denitrifying microorganisms is a problem to be carefully considered, namely, the reduction of nitrate ions in soil is required, the accumulation of excessive nitrite cannot be caused for a long time, the serious loss of soil nitrogen cannot be caused, and the stability of soil microecology is ensured. The algal polysaccharide serving as a biostimulator can promote the growth of denitrifying bacteria, meanwhile, the problems are avoided through the determination of relevant indexes, and the algal polysaccharide has a certain prospect when being used for stimulating the denitrifying bacteria to be used for rapidly improving the secondary salinization of soil.
The invention content is as follows:
the invention aims to provide a medicament and a method for promoting microorganisms to repair soil secondary salinization, which promote the growth of denitrifying bacteria and strengthen the capability of dissimilating nitrate nitrogen into ammonia through algal polysaccharides, and solve the problems of poor repairing effect and short survival time of strains when the existing microorganisms repair soil secondary salinization.
A medicament for promoting microorganism to restore soil secondary salinization mainly comprises denitrifying bacteria and algal polysaccharide.
A method for promoting microbial remediation of soil secondary salinization comprises spraying denitrifying bacteria suspension OD and algal polysaccharide solution into soil to be remedied600The concentration of the seaweed polysaccharide solution is 0.05 to 5 percent.
Specifically, the preparation method of the algal polysaccharide solution comprises the following steps: adding the dried seaweed powder into distilled water of 20-50 times, extracting for 40 minutes in a boiling water bath, removing excessive water by rotary evaporation, adding 95% ethanol of 4 times volume, precipitating with ethanol in a refrigerator at 4 ℃, centrifuging to obtain polysaccharide, dialyzing for two days by using a 3500Da dialysis bag after redissolving, freeze-drying after dialysis, weighing 1g of seaweed polysaccharide, and dissolving in 1L of distilled water to obtain the microbial accelerant.
Specifically, the preparation method of the denitrifying bacteria bacterial suspension comprises the following steps: activating denitrifying bacteria by using a solid culture medium, then scribing, placing a flat plate with a single colony in a refrigerator at 4 ℃ for storage, picking the single colony in the culture medium when in use, and then diluting after centrifugation to obtain a heavy suspension.
The algal polysaccharides include, but are not limited to, one or more of red algal polysaccharides, green algal polysaccharides, and brown algal polysaccharides. The molecular weight range is more than 3500 Da.
The denitrifying bacteria include but are not limited to Bacillus, Pseudomonas.
Compared with the prior art, the invention has the following beneficial effects: (1) the addition of a very small amount of algal polysaccharide can accelerate the growth of denitrifying bacteria, probably because monosaccharides, sulfate groups, carboxyl groups or complex structures in algal polysaccharide have a promoting effect on the growth of denitrifying bacteria; (2) the addition of the algal polysaccharide can be used for pertinently reducing the nitrate content in soil, and specifically shows that the nitrate ion consumption capacity is enhanced, the nitrite accumulation content is reduced, and the denitrifying bacteria dissimilatory reduction capacity is enhanced, which is probably because the addition of the algal polysaccharide can selectively stimulate related targets, so that bacteria are promoted to secrete corresponding enzymes, or a better reaction site can be provided for bacterial metabolism, so that secondary salinized soil can be quickly, efficiently and safely repaired; (3) the algal polysaccharide serving as a biological stimulator can promote plant growth and improve the stress resistance of plants, and meanwhile, rich organic matters can be provided for soil, the soil structure is improved, and the water and air permeability of the soil is improved; (4) the method is green, safe and capable of quickly relieving the problem of secondary salinization of soil.
Description of the drawings:
FIG. 1 is a graph showing the effect of the addition of algal polysaccharides on the growth of Bacillus in examples 1-3.
FIG. 2 is a graph of the effect of addition of algal polysaccharides on growth of Pseudomonas in examples 1-3.
FIG. 3 is a histogram of nitrate consumption in Bacillus treated groups after addition of algal polysaccharides in examples 1-3.
FIG. 4 is a bar graph of nitrate consumption in the Pseudomonas treatment group after addition of algal polysaccharides in examples 1-3.
FIG. 5 is a histogram of ammonia nitrogen accumulation in Bacillus treated groups after addition of algal polysaccharides in examples 1-3.
FIG. 6 is a histogram of ammonia nitrogen accumulation in the Pseudomonas group following addition of algal polysaccharides in examples 1-3.
FIG. 7 is a histogram of nitrite nitrogen accumulation in Bacillus treated groups after addition of algal polysaccharides in examples 1-3.
FIG. 8 is a histogram of nitrite nitrogen accumulation in the Pseudomonas group after addition of algal polysaccharides in examples 1-3.
FIG. 9 is a bar graph of the total nitrogen change in the Bacillus treated groups after addition of algal polysaccharides in examples 1-3.
FIG. 10 is a bar graph of the total nitrogen change in the Pseudomonas treated group after addition of algal polysaccharides in examples 1-3.
The specific implementation method comprises the following steps:
the present invention will be described in further detail below by way of examples, but the present invention is not limited to the following embodiments.
Example 1:
the method for promoting the microbial remediation of the secondary salinization of soil, which is related by the embodiment, comprises the following steps:
(1) firstly, adding dry red algae powder into distilled water of 40 times, extracting for 40 minutes in a boiling water bath, removing excessive water by rotary evaporation, adding 95% ethanol of 4 times volume, precipitating with ethanol in a refrigerator at 4 ℃, centrifuging to obtain polysaccharide, dialyzing for two days by using a 3500Da dialysis bag after redissolution, carrying out freeze-drying treatment after dialysis is finished, weighing 1g of freeze-dried red algae polysaccharide, adding the freeze-dried red algae polysaccharide into 1L of distilled water, dissolving in the boiling water bath, and cooling to obtain the algal polysaccharide promoter.
(2) Selecting single colonies of bacillus and pseudomonas in LB and beef extract peptone liquid culture media, respectively carrying out shake culture at 37 ℃, 30 ℃ and 120rpm overnight, taking out the bacterial liquid the next day, centrifuging at 8000rpm for five minutes to remove the culture medium, washing twice with sterile water, adding a proper amount of sterile water, and uniformly shaking to obtain the bacillus bacterial suspension and the pseudomonas bacterial suspension.
(3) And (3) after stones and weeds in the collected soil are picked out, putting the soil into a sterilization pot for sterilization, then placing the sterilized soil in a ventilation place for air drying, sieving the soil after air drying, and placing the soil in a plastic package bag for storage.
(4) Weighing a certain amount of air-dried soil in plastic pots, adjusting the water content of the soil to 60% with distilled water, then filling 1kg of soil in each pot, and spraying OD (origin-destination) on bacillus control group600Bacillus suspension 20ml ═ 1 and deionized water 10ml, pseudomonas control group sprayed with OD60020ml of pseudomonas suspension and 10ml of deionized water, after 20ml of bacillus suspension and 10ml of pseudomonas suspension are respectively sprayed on the bacillus treatment group and the pseudomonas treatment group, continuously spraying 10ml of 0.1% red algae polysaccharide solution, putting the treated mixture into a greenhouse sterilized in advance, taking samples once every two days, and taking samplesAnd (3) putting the obtained soil sample in a plastic package bag for storage, and supplementing the water of the soil in the pot according to the proportion.
(5) The retrieved fresh soil was measured as 1: adding 10 parts of the solution into a 1MKCL solution, oscillating for 30min in an oscillator at 25 ℃ and 180rpm, taking out the suspension, centrifuging, decoloring the supernatant, putting the decolored supernatant into a freezer at-80 ℃, and then uniformly measuring nitrate nitrogen, nitrite nitrogen, ammonia nitrogen and total nitrogen. Specifically, the content of nitrate ions is measured by adopting an ultraviolet spectrophotometry, the content of nitrite is measured by adopting an N- (1-naphthyl) -ethylenediamine photometric analysis method, the content of ammonia nitrogen is measured by adopting a salicylic acid-hypochlorite photometric method, and the total nitrogen content is measured by adopting a potassium persulfate oxidation-ultraviolet spectrophotometry method.
Example 2:
this example is the same as example 1 except that the algal powder in step (1) is green algal powder.
Example 3:
this example is the same as example 1 except that the seaweed powder in step (1) is brown seaweed powder.
Example 4:
this example is the same as example 1 except for the following steps.
(4) Weighing a certain amount of air-dried soil in plastic pots, adjusting the water content of the soil to 60% with distilled water, then filling 1kg of soil in each pot, and spraying OD (origin-destination) on bacillus control group600Bacillus suspension 30ml ═ 1 and deionized water 5ml, pseudomonas control group sprayed with OD60030ml of pseudomonas suspension and 5ml of deionized water, after 30ml of bacillus and pseudomonas suspension is sprayed on the bacillus treatment group and the pseudomonas treatment group respectively, 5ml of 1% rhodophyta polysaccharide solution is sprayed on the bacillus and pseudomonas suspension, the treated pseudomonas and rhodophyta polysaccharide solution are placed in a greenhouse sterilized in advance, samples are taken every two days, soil samples are obtained and placed in plastic package bags for storage, and the water content of the soil in the pot culture pot is supplemented according to the proportion.
Example 5:
this example is the same as example 1 except for the following steps.
(4) Weighing a certain amount of air-dried soil in a plastic basinIn the method, distilled water is used for adjusting the water content of soil to 60%, then 1kg of soil is filled in each pot, and OD is sprayed on a bacillus control group600Bacillus suspension 20ml ═ 1 and deionized water 20ml, pseudomonas control group sprayed with OD60020ml of pseudomonas suspension and 20ml of deionized water, 20ml of bacillus suspension and 20ml of pseudomonas suspension are sprayed on the bacillus treatment group and the pseudomonas treatment group respectively, 20ml of 5% rhodophyta polysaccharide solution is sprayed on the bacillus treatment group and the pseudomonas treatment group, the treated pseudomonas suspension and the bacillus treatment group are placed in a greenhouse sterilized in advance, samples are taken every two days, obtained soil samples are placed in plastic package bags for storage, and the water content of the soil in the pot culture pot is supplemented according to the proportion.
The experimental results show that: the addition of algal polysaccharides promotes the growth of bacillus and pseudomonas (see figure 1 and figure 2); secondly, the addition of algal polysaccharides accelerates the consumption of nitrate by denitrifying bacteria (see fig. 3 and 4); thirdly, the addition of algal polysaccharides improves the dissimilatory reduction capability of denitrifying bacteria, increases the accumulation of ammonia nitrogen in soil, reduces the conversion of nitrate ions to nitrogen, is favorable for balancing the balance of ammonia and nitrate nitrogen, and promotes the growth of plants (see fig. 5 and 6); the addition of algal polysaccharides can reduce the accumulation of nitrite ions in the denitrification process and reduce the potential hazard of soil denitrification by denitrifying bacteria (see fig. 7 and 8). The total nitrogen level of the soil is increased after the addition of the algal polysaccharides, which may be related to the secretion of extracellular substances of denitrifying bacteria, and is beneficial to improving the soil structure and improving the soil quality (see figure 9 and figure 10). The chlorella polysaccharide can better promote the growth of denitrifying bacteria and can also reduce the nitrate content more quickly.

Claims (6)

1. A method for promoting microorganisms to restore secondary salinization of soil is characterized in that denitrifying bacteria bacterial suspension and algal polysaccharide solution are sprayed into the soil to be restored.
2. The method for promoting microbial remediation of soil secondary salination of claim 1, wherein the algal polysaccharide is one or more of red algal polysaccharide, green algal polysaccharide and brown algal polysaccharide.
3. The method for promoting microbial remediation of soil secondary salination of claim 1, wherein the denitrifying bacteria is Bacillus, Pseudomonas.
4. The method for promoting microbial remediation of secondary salination of soil according to claim 1, wherein the algal polysaccharide solution is prepared by: adding the dried seaweed powder into distilled water of 20-50 times, extracting for 40 minutes in a boiling water bath, removing excessive water by rotary evaporation, adding 95% ethanol of 4 times volume, precipitating with ethanol in a refrigerator at 4 ℃, centrifuging to obtain polysaccharide, dialyzing for two days by using a 3500Da dialysis bag after redissolving, freeze-drying after dialysis, and weighing 1g of seaweed polysaccharide to be dissolved in 1L of distilled water.
5. The method for promoting microbial remediation of soil secondary salinization of claim 1, wherein the denitrifying bacteria bacterial suspension is prepared by the following steps: activating denitrifying bacteria by using a solid culture medium, then scribing, placing a flat plate with a single colony in a refrigerator at 4 ℃ for storage, picking the single colony in the culture medium when in use, and diluting after centrifugation to obtain a heavy suspension.
6. A medicament for promoting microorganisms to restore soil secondary salinization is characterized by comprising main components of denitrifying bacteria and algal polysaccharides.
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CN114467405A (en) * 2022-02-10 2022-05-13 自然资源部天津海水淡化与综合利用研究所 Method for restoring saline-alkali soil by culturing indigenous microorganisms and application thereof

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CN114467405A (en) * 2022-02-10 2022-05-13 自然资源部天津海水淡化与综合利用研究所 Method for restoring saline-alkali soil by culturing indigenous microorganisms and application thereof

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