CN113337420A - Composite nitrogen-fixing microbial agent and preparation method and application thereof - Google Patents
Composite nitrogen-fixing microbial agent and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganism application, and relates to a composite nitrogen-fixing microbial agent, and a preparation method and application thereof. The microbial agent is a mixed fermentation broth obtained by inoculating azotobacter, hydrogenophile and growth-promoting rhizobia in an improved BPY culture medium and performing liquid fermentation; the effective viable count of three single strains in the mixed fermentation liquor is not less than 1 multiplied by 106CFU/mL; the total effective viable count in the mixed fermentation liquor is not less than 1 × 109CFU/mL. The invention relates to composite nitrogen fixationThe microbial agent is based on the interaction relationship among azotobacter, growth-promoting rhizobium and hydrogenophile, the nitrogen fixation effect of the azotobacter is utilized to increase the nitrogen content of soil, and H is treated by the hydrogenophile2The reducing power degrades macromolecular organic matters, the nitrogen content of the soil is improved, the growth-promoting rhizobia is utilized to promote root systems of crops to generate root nodules, the nitrogen content of the soil is further increased, the three strains are compounded, extracellular polysaccharide is promoted to be generated by the crops, the volatile organic matters of the root communities are increased, the activity of original nitrogen-fixing bacteria in soil indigenous flora is improved, and the nitrogen fixation efficiency is comprehensively improved.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to a composite nitrogen-fixing microbial agent and a preparation method and application thereof.
Background
Nitrogen is one of the essential macro-elements in the crop growth process, and contributes about 40-50% to the final yield of crops. Crops take away a large amount of nitrogen from soil every year, and in order to compensate for the loss of the nitrogen in the soil, industrial chemical nitrogen fertilizers are still important sources for applying nitrogen to the soil by human beings. Although the fertilizer plays a great role in promoting plant growth and improving the yield of seeds, a series of problems such as acid deposition, soil compaction and nitrate accumulation in soil water can be caused by long-time excessive use of the fertilizer; global warming, cavities in odor layers and particulate matter formed by salt ions lead to photochemical smog and pollution of drinking water and food, and seriously hinder sustainable development of agriculture.
The nitrogen in the air can be converted into ammonia which can be used by plants through biological nitrogen fixation so as to meet the growth requirements of the plants. Compared with the application of chemical fertilizers, the biological nitrogen fixation does not need to consume non-renewable energy, the fixed nitrogen product can be directly absorbed and utilized by plants, and the agricultural environment pollution caused by volatilization, denitrification and leaching loss can be avoided. Therefore, the development of biological nitrogen fixation resources becomes a research hotspot in all countries of the world.
Biological nitrogen fixation comprises autogenous nitrogen fixation, joint solitary monomer and symbiotic nitrogen fixation, and Chinese scholars generally study rhizobia (symbiotic nitrogen fixation) in the coming years. Symbiotic nitrogen-fixing microorganisms are frequently studied, such as nitrogen-containing cyanobacteria feces (patent No.: ZL97117251.X), bradyrhizobium rhizogenes (patent No.: ZL200710032269.8), rhizobia DZY-N33 azotobacterin (patent No.: ZL200710032269.8), stenotrophomonas maltophilia (publication No.: 101781628A), and the like. Meanwhile, the nitrogen-fixing rhizobia and rhizobia fertilizer are only applied to low-yield fields or new growing areas, and the effect is obvious. The research and test on the relationship between bacteria are not perfect, the microbial inoculum is single, and the cooperation between bacteria is lacked.
The existing microbial nitrogen-fixing microbial inoculum in the market is similar in pesticide effect, short in pesticide effect duration, only suitable for a specific crop and narrow in application range, is lack of supplement to a carbon source, and a large number of symbiotic nitrogen-fixing bacteria researched in the past are not suitable for being applied to gramineae grain crops and non-leguminous vegetable crops.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a synergistic microbial agent and a preparation method and application thereof.
Based on the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a compound nitrogen-fixing microbial agent, which is a mixed fermentation broth obtained by inoculating nitrogen-fixing bacteria, hydrogenophiles and growth-promoting rhizobia into an improved BPY culture medium and performing liquid fermentation; the effective viable count of the three single strains in the mixed fermentation liquor is not less than 1 multiplied by 106CFU/mL; the total effective viable count in the mixed fermentation liquor is not less than 1 × 109CFU/mL。
The composite nitrogen-fixing microbial agent provided by the invention is based on the interaction relationship among azotobacter, growth-promoting rhizobium and hydrogenophile, the nitrogen-fixing effect of azotobacter is utilized to increase the nitrogen content of soil, and the hydrogenophile is utilized to carry out H treatment on H2Reducing power assimilation macromolecule difficult decomposition organic matter further promotes soil nitrogen content, promotes soil organic matter content simultaneously, can also impel the crop to produce rhizobium and further increases soil nitrogen content, and in addition, the benign interaction of three fungus promotes the crop to produce extracellular polysaccharide, rhizosphere volatile organic matter, promotes the activity of original azotobacter in the soil indigenous flora, synthesizes promotion fixed nitrogen efficiency.
The composite nitrogen-fixing microbial agent provided by the invention has the effects of promoting the growth of plant vascular tissues, thickening parenchyma cells, increasing the content of soluble sugar and nitrogen of plants, increasing the dry weight of overground parts of plants and comprehensively promoting the growth of plants.
The composite nitrogen-fixing microbial agent provided by the invention can be colonized in soil for a long time, so that the microbial diversity of the soil is increased, macromolecular organic matters in the soil are decomposed, the organic matter content in the soil is increased, the pH value of the soil is improved, and the acidification or alkalization of the soil is improved.
Furthermore, in the total viable count of the mixed fermentation liquor, the ratio of the viable count of the azotobacter to the viable count of the hydrogenophilic bacteria is 10-40%, and the ratio of the viable count of the hydrogenophilic bacteria is 20-50%; the active bacteria of the growth-promoting rhizobia accounts for 10 to 40 percent.
Further, the Azotobacter is at least one of Azospirillum brasiliensis (Azospirillum brasilense), Azospirillum melini (Azospirillum melinis) and Azospirillum chroococcum (Azotobacter chroococcum); the hydrogenophile is at least one of a hydrogenophile atypical (Hydrogenophaga typica), a hydrogenophile flavum (Hydrogenophaga flava), a hydrogenophile lisobospora lescens (Hydrogenophaga laconensis), a Pseudomonas pseudoxanthophylla (Hydrogenophaga pseudolava), a hydrogenophile Sewage hydrogenophile (Hydrogenophaga defluvii) and a hydrogenophile geophilum (Hydrogenophaga soli); the growth promoting rhizobia is at least one of Lactobacilli cinerea (Shinella zoogloeoides), Stachybotrys helophora (Shinella kummerowiae), and Stachybotrys graminearus (Shinella grandifolia).
Further, the azotobacter is Azospirillum brasiliensis (Azospirillum brasilense); the hydrogenophile is atypical hydrogenophile spore (Hydrogenophaga typica); the root nodule bacteria is Acinetobacter pseudostellatus (Shinella zoogloeoides).
Further, Azospirillum brasiliensis (Azospirillum brasilense) is a strain with the number of CGMCC: 1.10379; the atypical hydrogenophilus (Hydrogenophaga atypica) is a strain with the number of CGMCC: 1.13009; the animal glue fungus-like Shen bacterium (Shinella zoogloeoides) is the strain with the number of CGMCC: 1.6838.
Further, the azotobacter is Azospirillum brasiliensis (Azospirillum brasilense); the hydrogenophilic bacteria is hydrogen-philospora raphanus (Hydrogenophaga laconesensis); the root nodule bacteria is Acinetobacter pseudostellatus (Shinella zoogloeoides).
Further, the azotobacter is Azospirillum melitensis (Azospirillum melinis); the hydrogenophilic bacteria is hydrogen-philospora raphanus (Hydrogenophaga laconesensis); the growth promoting rhizobium is Acremonium gallinarum (Shinella kummelowiae).
Further, the azotobacter is Azospirillum melitensis (Azospirillum melinis); the hydrogenophile is atypical hydrogenophile spore (Hydrogenophaga typica); the growth promoting rhizobium is Acremonium gallinarum (Shinella kummelowiae).
Screening 81 groups of strains with different combinations in azotobacter, growth-promoting rhizobia and hydrogenophilus, and finally screening out the four groups of microbial inoculum with good crop growth promotion effect.
Further, the improved BPY culture medium takes water as a medium and comprises the following components: 3.75-6.25 g/L peptone, 1.13-1.88 g/L beef extract, 7.50-12.5 g/L sucrose, 0.12-0.19 g/L yeast extract, 1.88-3.13 g/L NaCl, 0.2g/L KH2PO4、0.8g/L K2HPO40.075-0.125 g/L magnesium sulfate heptahydrate, 0.038-0.063 g/L calcium sulfate dihydrate, 0.00005-0.0005 g/L ferric chloride, 0.00005-0.0005 g/L sodium tungstate, 0.00005-0.0005 g/L sodium molybdate and 0.00005-0.0005 g/L manganese sulfate.
The KH is further increased on the basis of the existing BPY culture medium by the improved BPY culture medium2PO4And K2HPO4As a pH buffer pair, the pH stability of the culture solution is ensured; magnesium sulfate and calcium sulfate are added as mineral elements on which the nitrogen-fixing bacteria live; four trace elements of iron, tungsten, molybdenum and manganese are added to promote the growth of azotobacter; meanwhile, the contents of peptone, beef extract, yeast extract and sodium chloride are reduced to control the concentration of nutrient substances, so that the improved BPY culture medium can effectively ensure the coexistence of azotobacter, hydrogenophile and growth-promoting rhizobia in the same system.
The adjustment enables the improved BPY culture medium to be suitable for mixed culture of azotobacter, rhizobium growth-promoting bacteria and hydrogenophile, and enables the three strains to have higher active bacteria number after mixed culture.
In a second aspect, the invention provides a production method of the composite nitrogen-fixing microbial agent, which comprises the following steps:
(1) the azotobacteria, the hydrogenophilous bacteria and the growth-promoting rhizobia are respectively cultured in corresponding culture media separately to respectively prepare azotobacteria for fermentationLiquid, hydrogenophilic bacteria fermentation liquid and growth promoting rhizobium fermentation liquid: the viable count of nitrogen-fixing bacteria, hydrogenophilic bacteria and growth-promoting rhizobia in nitrogen-fixing bacteria fermentation liquor, hydrogenophilic bacteria fermentation liquor and growth-promoting rhizobia fermentation liquor is not less than 2 multiplied by 108CFU/mL；
(2) Equivalently adding the nitrogen-fixing fermentation broth, the hydrogenophile fermentation broth and the growth-promoting rhizobium fermentation broth prepared in the step (1) into an improved BPY culture medium, and fermenting for 24-48 h at 26-30 ℃ and 150-200 rpm/min to prepare a mixed fermentation broth, wherein the viable count of the three single strains in the mixed fermentation broth is not less than 1 x 106CFU/mL; the total viable count in the mixed fermentation liquid is not less than 1 × 109CFU/mL。
Further, the nitrogen-fixing bacteria culture medium takes water as a medium and comprises the following components: 0.375-0.625 g/L yeast extract, 15-25 g/L mannose, 0.2g/L monopotassium phosphate, 0.8g/L dipotassium phosphate, 0.15-0.25 g/L magnesium sulfate heptahydrate, 0.075-0.125 g/L calcium sulfate, 0.00005-0.0005 g/L ferric chloride and 0.00005-0.0005 g/L sodium molybdate;
the hydrogenophilic bacteria culture medium takes water as a medium and comprises the following components: 7.5-12.5 g/L of peptone, 2.25-3.75 g/L of beef extract and 3.75-6.25 g/L of NaCl;
the growth promoting rhizobium culture medium takes water as a medium and comprises the following components: 7.5-12.5 g/L peptone, 2.25-3.75 g/L beef extract and 3.75-6.25 g/L NaCl.
The azotobacter, the hydrogenophilic bacteria and the growth-promoting rhizobia cultured by the improved BPY culture medium have higher viable count.
In a third aspect, the composite nitrogen-fixing microbial agent is applied to the rejuvenation of soybean nitrogen fixing, peanut, wheat, oat and corn.
In a fourth aspect, the composite nitrogen fixation microbial agent disclosed by the invention is applied to relieving soil acid basification.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention is based on the interaction relation of azotobacter, hydrogenophile and growth-promoting rhizobium, utilizes the nitrogen fixation effect of azotobacter to increase the nitrogen content of soil, and uses hydrogenophile to H2Reducing power of (2)The organic matter is used for increasing the nitrogen content of the soil, the growth-promoting rhizobia is used for promoting root systems of crops to generate rhizobia so as to further increase the nitrogen content of the soil, the compounding of the three strains is beneficial to improving the activity of the original nitrogen-fixing bacteria in the soil indigenous flora, and the nitrogen-fixing capability of the composite nitrogen-fixing microbial agent prepared from the three strains is comprehensively improved; the composite nitrogen-fixing microbial agent can promote the growth of plant vascular tissues and the thickening of parenchyma cells, increase the content of soluble sugar and nitrogen of plants and comprehensively promote the growth of plants.
(2) The composite nitrogen-fixing microbial agent can be colonized in a plant rhizosphere for a long time, the activity of soil indigenous nitrogen-fixing bacteria is enhanced, and meanwhile, the diversity of soil microorganisms is increased.
(3) The composite nitrogen-fixing microbial agent can decompose macromolecular organic matters which are difficult to be directly utilized by plants in soil into micromolecular organic matters, improve the nitrogen content of the soil, improve the pH value of the soil and improve the acidification or alkalization of the soil.
In conclusion, the composite nitrogen-fixing microbial agent disclosed by the invention can enhance the nitrogen-fixing effect, promote the growth of crops, effectively improve the pH value of soil and improve the microbial diversity of the soil.
Drawings
FIG. 1 is a microscopic examination of azotobacteria;
FIG. 2 is a microscopic examination of nodule-promoting bacteria;
FIG. 3 is a microscopic examination of hydrogenophiles;
FIG. 4 is a colony map of a nitrogen-fixing bacteria plate;
FIG. 5 is a colony map of a growth-promoting rhizobium plate;
FIG. 6 is a colony diagram of a hydrogenophile plate;
FIG. 7 is a microscopic examination of mixed bacteria of azotobacteria, rhizobium growth promoting bacteria and hydrogenophilum;
FIG. 8 is a plate colony image of microorganisms in soil at the start of the test in example 3;
FIG. 9 is a plate colony image of microorganisms in soil after two months of the test in example 3;
FIG. 10 is a paraffin section of peanut caulicle tissue.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used can be obtained from commercial approaches if no special description exists, the tested strains can be obtained from commercial approaches, such as the strains can be purchased from strain preservation centers of CGMCC, CICC, ATCC and the like, and the sources of the strains except the optimal tested strains are not repeated.
Example 1 preparation of composite Nitrogen-fixing microbial Agents
The preparation of the composite nitrogen-fixing microbial agent mainly comprises the following steps:
1. individual culture of microorganisms
(1) Independent culture of azotobacteria
Obtaining azotobacter powder or bacterial liquid or a test tube inclined plane from a commercial channel, placing an azotobacter sample into 0.3mL azotobacter fermentation liquid to obtain a recovered azotobacter suspension, adding the recovered azotobacter suspension or bacterial colony into the azotobacter fermentation liquid as seed liquid, inoculating azotobacter strain into fermentation liquid with pH of 7.2 according to the proportion of adding 0.3mL seed liquid into every 100mL fermentation liquid, and fermenting for 24-48 h under the conditions of 26-30 ℃ and 150-200 rpm/min until the effective viable count of azotobacter is more than 2 multiplied by 108CFU/mL to obtain the azotobacter culture solution. Wherein the azotobacter is Azospirillum brasilense.
The fermentation liquor for culturing the azotobacter strain comprises the following specific components:
0.5g yeast extract, 20.0g mannitol, 0.2g potassium dihydrogen phosphate, 0.8g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.1g calcium sulfate, 0.0005g ferric chloride, 0.0005g sodium molybdate, and 1L distilled water.
Wherein the Azotobacter can be at least one of Azospirillum melini (Azospirillum melinis) and Azospirillum chroococcum (Azotobacter chroococcum).
(2) Individual culture of growth-promoting rhizobia
Obtaining growth-promoting rhizobia powder or bacterial liquid or a test tube inclined plane from a commercial channel, placing a growth-promoting rhizobia sample in 0.3mL of growth-promoting rhizobia fermentation liquor to obtain a suspension of revitalized growth-promoting rhizobia, adding the revitalized growth-promoting rhizobia suspension or bacterial colony serving as seed liquid into the growth-promoting rhizobia fermentation liquor, inoculating the growth-promoting rhizobia strain into the fermentation liquor with the pH of 7.0 according to the proportion of adding 0.3mL of seed liquid into every 100mL of fermentation liquor, and fermenting for 24-48 hours under the conditions of 26-30 ℃ and 150-200 rpm/min until the effective viable count of the growth-promoting rhizobia is more than 2 x 108CFU/mL to obtain the growth-promoting rhizobium culture solution. Wherein the rhizobium growth-promoting bacteria is Proteus zoogloeoides (Shinella zogloeoides).
The fermentation liquor for culturing the rhizobium growth-promoting strain comprises the following specific components:
10.0g peptone, 3.0g beef extract, 5.0g sodium chloride, 1L distilled water.
Wherein the rhizobium growth-promoting bacteria can also be at least one of Schneisseria gallinarum (Shinella kummerowiae) and Schneisseria graminis (Shinella grandis).
(3) Independent culture of hydrogenophiles
Obtaining hydrogenophile powder or bacterial liquid or a test tube inclined plane from a commercial channel, placing a hydrogenophile sample into 0.3mL of hydrogenophile fermentation liquor to obtain a resuscitated hydrogenophile suspension, adding the resuscitated hydrogenophile suspension or bacterial colony into the hydrogenophile fermentation liquor as seed liquor, inoculating hydrogenophile seeds into the fermentation liquor with the pH of 7.0 according to the proportion of adding 0.3mL of seed liquor into every 100mL, and fermenting for 24-48 h under the conditions of 26-30 ℃ and 150-200 rpm/min until the effective viable count of the hydrogenophile in the fermentation liquor is more than 2 x 108CFU/mL to obtain a hydrogenophile culture solution. Among them, hydrogenophiles are atypical hydrogenophiles (hydrogenophoga atypica).
The fermentation liquor for culturing the hydrogenophiles comprises the following specific components:
10.0g peptone, 3.0g beef extract, 5.0g sodium chloride, 1L distilled water.
The hydrogenophile may be at least one of hydrogenophile chrysosporium (hydrogenophila flava), hydrogenophile laishen (hydrogenophila laconensis), pseudohydrogenophile chrysosporium (hydrogenophila pseudo-lava), hydrogenophile sewerae (hydrogenophila defluvivii), and hydrogenophile geotrichum (hydrogenophila soli).
0.3mL of the azotobacter culture solution, the growth-promoting rhizobium culture solution and the hydrogenophile culture solution are respectively dripped on a glass slide, and are dried and microscopically examined as shown in figures 1-3.
And (3) respectively scribing the azotobacter culture solution, the growth-promoting rhizobium culture solution and the hydrogenophile culture solution by using 10 mu L inoculating loop plates, and recording after bacterial colonies grow out, wherein plate bacterial colony maps of the three bacterial strains are respectively shown in fig. 4-6.
2. Mixed culture of microbiome
Inoculating the azotobacteria culture solution, the growth promoting rhizobium culture solution and the hydrogenophile culture solution obtained in the step 1 into an improved BPY culture medium according to the proportion of adding 0.25mL into each 100mL of the improved BPY culture medium, and fermenting for 24-48 h at 26-28 ℃ and 150-200 rpm/min until the total effective viable count in the mixed fermentation liquid is more than 1 × 109CFU/mL, the effective viable count of azotobacteria, rhizobium growth-promoting bacteria and hydrogenophilum in the mixed fermentation liquor is not less than 1 x 106And CFU/mL to prepare the composite nitrogen-fixing microbial agent.
The mixed fermentation solution of 0.3mL is dropped on a glass slide, and dried and examined under a microscope as shown in FIG. 7.
In the total viable count of the mixed fermentation liquor, the ratio of the viable count of the azotobacter to the viable count of the hydrogenophilic bacteria is 10-40 percent, and the ratio of the viable count of the hydrogenophilic bacteria to the viable count of the hydrogenophilic bacteria is 20-50 percent; the active bacteria of the growth-promoting rhizobia accounts for 10 to 40 percent.
The formula of the improved BPY culture medium is shown in Table 1, and the components and the using amount of the existing BPY culture medium are adjusted, so that the improved BPY culture medium provided by the invention can be simultaneously suitable for culturing azotobacter, rhizobium growth-promoting bacteria and hydrogenophile, and the three bacteria have higher effective viable count after mixed culture.
Based on the existing BPY culture medium, the improved BPY culture medium provided by the invention adds dipotassium hydrogen phosphate and potassium dihydrogen phosphate as buffer solution pairs, so that the pH stability of the culture solution is ensured; magnesium sulfate and calcium sulfate are added as mineral elements on which the nitrogen-fixing bacteria live; four trace elements of iron, tungsten, molybdenum and manganese are added to promote the growth of azotobacter; meanwhile, the contents of peptone, beef extract, yeast extract and sodium chloride are reduced to control the concentration of nutrient substances, so that the improved BPY culture medium can effectively ensure the coexistence of azotobacter, hydrogenophile and growth-promoting rhizobia in the same system.
TABLE 1 formulation of modified BPY Medium
| Components | Dosage of | Components | Dosage of |
| Peptone | 5.0g | Beef extract | 1.5g |
| Sucrose | 10.0g | Yeast extract | 0.15g |
| NaCl | 2.5g | KH2PO4 | 0.2g |
| K2HPO4 | 0.8g | Magnesium sulfate heptahydrate | 0.1g |
| Calcium sulfate dihydrate | 0.05g | Ferric chloride | 0.0005 |
| Sodium tungstate | 0.0005 | Sodium molybdate | 0.0005 |
| Manganese sulfate | 0.0005 | Water (W) | 1L |
Example 2 Performance testing of composite Nitrogen-fixing microbial Agents
In this example, the performance of the composite nitrogen-fixing microbial agent prepared in example 1 is tested in the following four aspects.
1. Hemolytic reaction of microbial agent
The mixed fermentation liquid prepared in the example 1 is inoculated on a blood agar plate and cultured for 24 hours at 37 ℃, and no hemolytic cycle is observed to be generated, which shows that the composite nitrogen-fixing microbial agent prepared by the invention can be used as an agricultural microbial agent.
The blood agar plate formulation in the experiment was as follows: 10g of peptone, 10g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of distilled water, wherein the pH value is 7.5, the beef extract powder is sterilized under high pressure and cooled to 60 ℃, sterile defibrinated sheep blood is added, the mixture is fully rotated and poured into a flat plate, and the thickness of the blood agar layer is 5 mm.
2. Detection of hydrogen production capability of microbial agent
The azotobacteria culture solution, the hydrogenophile culture solution and the mixed fermentation liquid in the example 1 are respectively mixed with LB agar to prepare slant culture media (note: no test is carried out because a single strain of the growth-promoting rhizobia does not have the capability of producing hydrogen), 5mL of the slant culture media are added into a test tube with the specification of 15mm x 150mm, the test tube is sealed by a soft rubber plug for culture, the test tube is kept still for 24H until bacterial lawn grows out, 5mL of gas is extracted, 5mL of hydrogen is added, 100 muL of gas in the test tube is extracted after 48H of culture, the hydrogen consumption (namely the hydrogen absorption rate) is measured by a gas chromatograph, the test results are shown in a table 2, the negative value in the table is H, H is shown in a negative value2The results in table 2 show that the composite nitrogen-fixing microbial agent prepared by the invention has the highest hydrogen production capacity compared with any single bacterial strain, and the results show that the composite nitrogen-fixing microbial agent prepared by the invention has good hydrogen production capacity.
The LB agar used in the test is formulated as follows: 10g of peptone, 5g of yeast extract, 5g of sodium chloride and 15g of agar.
TABLE 2 different microbial inoculum pairs H2Absorption rate results of
| Test group of species of strains | H2Absorption rate (%) |
| Azotobacteria, growth-promoting rhizobia and hydrogenophiles | -6.14 |
| Hydrogen-philic bacterium | 45.01 |
| Azotobacteria | -34.49 |
3. Detection of nitrogen fixation capability of microbial agent
The azotobacteria culture solution, the hydrogenophila culture solution and the mixed fermentation liquid in the example 1 are respectively mixed with LB agar to prepare slant culture media, meanwhile, using equal amount of clear water as a reference, adding 5mL of slant culture medium into a test tube with the specification of 15mm x 150mm, sealing with a soft rubber plug, culturing, standing for 24 hr, collecting 5mL air, injecting 5mL acetylene, culturing for 48 hr, collecting 100 μ L gas in the test tube, measuring ethylene production with gas chromatograph, the nmol amount of acetylene produced by each mg of bacterial liquid per hour is used for evaluating the nitrogen fixing capacity of each strain, the higher the ethylene production amount is, the stronger the nitrogen fixing capacity of the microbial agent is, the test result is shown in table 3, the mean value of the nitrogen fixing capacity of the composite nitrogen-fixing microbial agent is obviously higher than the nitrogen fixing capacity value of other single strains, the result shows that the composite nitrogen-fixing microbial agent has more excellent nitrogen-fixing capacity compared with any single strain.
TABLE 3 Nitrogen fixation Capacity values of different bacterial agents
| Test group of species of strains | Mean nitrogen fixation (nmolC)2H4) |
| Azotobacteria, growth-promoting rhizobia and hydrogenophiles | 7854 |
| Hydrogen-philic bacterium | 0 |
| Azotobacteria | 3145 |
| Clear water control | 0 |
4. Detection of decomposition capability of microbial agent on macromolecular organic matter
The consumption of biochemical oxygen demand of the microbial inoculum related to the embodiment 1 is detected by adopting a five-day culture method, and meanwhile, the same amount of clear water is used as a reference, the specific method is the conventional method, which is not described herein, and the BOD of the sewage after bacteria filtration is used for detection5The stock reflects the decomposition capability, BOD, of different microbial inoculum to macromolecular organic matter5The lower the stock, the stronger the decomposition capability of the microbial agent on macromolecular organic matters, and the detection results are shown in Table 4, which shows that BOD after the composite nitrogen-fixing microbial agent treatment5The lowest stock shows that the composite nitrogen-fixing microbial agent formed by combining azotobacter, growth-promoting rhizobium and hydrogenophile has better decomposition capability of macromolecular organic matters compared with any single microbial agent.
TABLE 4 determination results of the decomposition ability of various bacteria to macromolecular organic substances
| Test group of species of strains | BOD5Inventory (mg/L) |
| Azotobacteria, growth-promoting rhizobia and hydrogenophiles | 173 |
| Hydrogen-philic bacterium | 191 |
| Azotobacteria | 198 |
| Control | 200 |
Example 3 interaction test of composite Nitrogen-fixing microbial Agents with plants
The embodiment researches the interaction relationship between the compound nitrogen-fixing microbial agent and the plant from two aspects of the colonization condition of the microorganism in the plant root ring environment and the influence on the plant tissue.
1. Colonization of microorganisms in the environment of plant rhizosphere
The crops are potted in an incubator, and soil for potting is humus and is from Changbai mountain in Jilin province. The experimental culture pot is 21cm x 21cm, and the physical and chemical properties of the soil are as follows: the average particle size in dry state was 223 μm, the particle size in wet state was 66.5 μm, and the particle volume was 4.64 x 105cm3The density of the dry humus soil was about 2.47g/cm 3. The mass fraction of the organic matters is 29.13 percent, the solid content is 89.50 percent, and the planting plant is peanut. The nitrogen-fixing microbial agent prepared in example 1 was diluted 25 times, and then 4 plants per pot were inoculated into a culture bowl, and the microbial agent diluted 100 times was sprayed to the leaf surface and root every 2 weeks. Bacteria were isolated in the soil rhizosphere environment after 2 months.
The microorganism in the soil is separated and coated on the flat plate respectively at the beginning of the test, as shown in fig. 8, after the test is carried out for two months, the microorganism in the soil is separated and coated on the flat plate, as shown in fig. 9, the content of each component of the microorganism group is estimated by adopting a flat plate counting method, and the result is shown in table 5.
TABLE 5 viable counts of azotobacter, growth-promoting rhizobia and hydrogenophilus in soil after 2 months of testing
| Bacterial strain | Viable count (CFU/g) |
| Azotobacteria | 8.9×107 |
| Growth promoting rhizobia | 2.6×106 |
| Hydrogen-philic bacterium | 2.1×107 |
2. Effect of composite nitrogen-fixing microbial agent on plant tissues
A stem plant tissue section experiment is carried out on crops planted in a culture pot for 1 month in an incubator environment, and the specific test method is as follows.
The incubator environment is: the light is 14 hours, the dark is 10 hours, the light intensity is 16000Lux, the temperature is 28 ℃, and the humidity is 75%.
The planting management scheme is as follows: the test group adopts the compound nitrogen-fixing microbial inoculum to soak the seeds, the peanut seeds are soaked for 1h at 28 ℃ by the microbial inoculum diluted by 25 times, the peanut seeds are planted into a culture bowl after germination acceleration, and the foliage is sprayed while the peanut seeds are irrigated once every two weeks by the microbial inoculum diluted by 100 times; the control group used a similar management scheme, but the inoculum was replaced with an equal amount of distilled water.
The stems of the plants planted in the test groups for 1 month were sliced, and as shown in fig. 10, the stem phloem tissue was thickened, the xylem was developed, the vascular tissue was developed, and the air permeability was increased.
3. Influence of composite nitrogen-fixing microbial agent on content of soluble sugar in plants
(1) Establishing a standard curve of absorbance-sugar content
The specific steps for establishing the standard curve of absorbance-sugar content are as follows: taking 6 test tubes with 20mL scales, numbering 1-6 in sequence, adding 100 mu g/mL sucrose solutions with the sugar contents of 0 mu g/mL, 10 mu g/mL, 20 mu g/mL, 30 mu g/mL, 40 mu g/mL and 50 mu g/mL into the 6 test tubes respectively, supplementing the solutions with water to 2mL, adding 0.5mL anthrone ethyl acetate reagent and 5mL concentrated sulfuric acid into each test tube in sequence, fully oscillating, immediately putting the test tubes into a boiling water bath, accurately preserving the temperature for 1min tube by tube, taking out, naturally cooling to room temperature to obtain a blank control, measuring the absorbance at a wavelength of 630nm, taking the absorbance as a vertical coordinate, taking the sugar content as a horizontal coordinate, drawing a standard curve, and calculating a standard linear equation.
(2) Sugar content extraction and detection
Taking peanut plants planted for 1 month in a test group and a control group with equal plant number and influence of 2 and compound nitrogen-fixing microbial agents on plant tissues, drying and crushing, accurately weighing 0.10-0.30 g of crushed samples, putting the crushed samples into a 20mL graduated test tube, adding 5-10 mL of distilled water, sealing by a plastic film, extracting in boiling water for 30min for 2 times, filtering the extracting solution into a 25mL volumetric flask, repeatedly washing the test tube and residues, and fixing the volume to the graduation to obtain a sample solution.
Sucking 0.5mL of sample liquid into a 20mL graduated test tube, adding 1.5mL of distilled water, sequentially adding an anthrone ethyl acetate reagent and a concentrated sulfuric acid solution according to the step of preparing a standard curve, developing, measuring absorbance, and calculating the sugar content in the test sample according to the standard curve. The results are shown in table 6, and compared with the control group, the soluble sugar content of the peanut plants treated by the composite nitrogen-fixing microbial agent is improved by 1.8 times, which shows that the composite nitrogen-fixing microbial agent can promote the growth of peanuts.
TABLE 6 sugar content of the differently treated plants
4. Influence of composite nitrogen-fixing microbial agent on nitrogen content of plants
Taking soybean seeds soaked for 20min by the compound microbial agent diluted by 50 times as an experimental group, and taking soybean seeds soaked for 20min by the culture solution with the same dilution times as a control group; the sampling place of the soybean seeds is a clear city district of Qingyuan, Guangdong province; the growing environment is a plant incubator. The grain protein content was measured with a portable soy protein determinator (FOSS infra 1241).
The content of the soybean seed protein treated by different treatments is shown in table 7, and the content of the soybean seed protein treated by the composite nitrogen-fixing microbial agent is higher than that of a control group, so that the microbial agent has the effect of increasing the content of the soybean seed protein.
TABLE 7 Soybean seed protein content of different treatments
| Processing method | Content of protein in Soybean seed (%) |
| Test set (composite nitrogen fixation microbial inoculum) | 47.1％ |
| Control group (distilled water) | 43.7％ |
Example 4 test of improving effects of composite nitrogen-fixing microbial agent on soil acidification and alkalization
In this embodiment, a test for investigating the improvement effect of the composite nitrogen-fixing microbial agent prepared in embodiment 1 on soil acidification and alkalization is performed, and the specific test method is as follows:
taking Guangdong Qingyuan red soil, Changbai mountain humus soil and pond bottom soil as test soil, sampling 10kg of each soil sample, adding 100mL of composite nitrogen-fixing microbial agent, mixing and stirring uniformly, putting into a constant-temperature constant-humidity incubator for culture, and sampling and detecting the pH value of the culture in 1d, 7d and 28d of culture respectively.
The sampling detection method comprises the following specific steps: respectively sampling 100g of test soil cultured in an incubator, adding the test soil into a beaker, adding distilled water to a constant volume of 1L, uniformly mixing the test soil with a vortex stirrer, and standing for 10 min; and (4) taking 100mL of supernatant, pouring into a triangular flask, measuring the pH by using a pH measuring instrument, and repeating the operation process for three times to obtain an average value.
The measurement results are shown in table 8, and it can be seen that after being treated by the microbial agent of the present invention, the pH values of the acidic laterite and humus soil are increased, and the pH values of the alkaline pond bottom soil are decreased, which indicates that the composite nitrogen-fixing microbial agent of the present invention has the effects of improving the pH of the soil, and improving the acidification and alkalization of the soil.
TABLE 8 soil pH values of 1d, 7d and 28d soil treatment by composite nitrogen-fixing microbial agent
Example 5 field test
The test field is located in the Guangdong Qingyuan Xinxin area, and the test crops comprise corn, wheat, oat, rape and peanut. The specific operation is as follows: crop seeds with uniform size, glossiness and similar shape are taken and soaked in a test mixed bacterial sample diluted by 50 times, and each crop is provided with 5 plants in one group. Within 1 month after sowing, the planting site was irrigated with 100-fold diluted mixed bacterial samples tested every 2 weeks. In the second February after sowing, the leaf spraying is carried out on the crops by using the tested mixed bacterial sample diluted by 100 times every 2 weeks. Collecting the overground part of the whole plant after planting for 2 months, drying to constant weight, measuring the dry weight growth rate of the overground part, and taking the average value of the growth rates of the crops of corn, wheat, oat, rape and peanut species as the final growth rate. The control group was treated with the corresponding clear water.
The test bacterial groups were combined as follows:
the tested azotobacter: azospirillum brasilense (Azospirillum brasilense), Azospirillum melini (Azospirillum melinis), Azospirillum irakense (Azospirillum irakense), Azospirillum halothrin (Azotobacter salinistis), Amazospirillum amazonensis (Azospirillum amazonense), Azospirillum torum chroococcum (Azotobacter chroococcum), Azospirillum lipolyticum (Azospirillum lipoferum), Azospirillum nidum (Azospirillum wenxinae), Azospirillum harderium (Azospirillum palatum) 9 in total.
Test hydrogenophiles: a total of 9 species of Hydrophilospora typicala (Hydrogenophaga typica), Hydrogenophycea flava (Hydrogenophaga flava), Hydrogenophycea leinshi (Hydrogenophaga laconensis), Hydrogenophycea pseudoflava (Hydrogenophaga pseudoflulava), Hydrogenophycea sewerae (Hydrogenophaga defluviii), Hydrogenophycea (Hydrogenophaga soli), Hydrogenophycea tenuii (Hydrogenhaga taeniiospira), Hydrogenophycea intermedia (Hydrogenhaga intermedium), and Hydrogenophycea palusta (Hydrogenhaga pallonii).
Test growth promoting rhizobia: 3 species of the species Shinella zoogloeoides (Shinella zoogloeoides), Shinella clavata (Shinella kummerowiae) and Shinella clavata (Shinella grandis).
The total 243 experimental groups and 1 control group are arranged and combined. Depending on the experimental design and analysis principles, the three-factor test can be projected as a two-factor test. The two-factor test has 81 experimental groups.
The test results of the 81 test groups on the average of the growth rates of crops of corn, wheat, oat, rape and peanut species are shown in table 9, and it can be seen that when the microbial agent prepared by combining azospirillum brasilense, alexandrium zoosum and atypical hydrogenophilus sporula or the microbial agent prepared by combining azospirillum brasilense, alexandrium zoosum and lance hydrogenophilus sporula has an excellent effect of promoting the growth of crops; and the second is a microbial agent prepared by combining azospirillum meliloti, aristolochia gallica and hydrogenophiles of the laishen, or a microbial agent prepared by combining azospirillum meliloti, aristolochia gallica and atypical hydrogenophiles.
TABLE 9 mean growth rates of crops treated with different microbial agents
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The compound nitrogen-fixing microbial agent is characterized in that the microbial agent is a mixed fermentation liquid obtained by inoculating nitrogen-fixing bacteria, hydrogenophiles and growth-promoting rhizobia to an improved BPY culture medium and performing liquid fermentation; the effective viable count of the three single strains in the mixed fermentation liquid is not less than 1 multiplied by 106CFU/mL; the total effective viable count in the mixed fermentation liquor is not less than 1 multiplied by 109CFU/mL。
2. The composite nitrogen-fixing microbial agent as claimed in claim 1, wherein in the total viable count of the mixed fermentation broth, the viable count of nitrogen-fixing bacteria accounts for 10% -40%, and the viable count of hydrogenophilic bacteria accounts for 20% -50%; the active bacteria of the growth-promoting rhizobia accounts for 10 to 40 percent.
3. The composite nitrogen-fixing microbial agent according to claim 1, wherein the nitrogen-fixing bacteria is at least one of Azospirillum brasiliensis (Azospirillum brasilense), Azospirillum melinii (Azospirillum melinis) and Azospirillum torvum (Azotobacter chroococcum); the hydrogenophile is at least one of atypical hydrogenophile spore bacteria (Hydrogenophaga typica), hydrogenophile yellow spore bacteria (Hydrogenophaga flava), hydrogenophile lainshii (Hydrogenophaga laconensis), pseudohydrogenophile flava spore bacteria (Hydrogenophaga pseudo-lava), hydrogenophile sewage spore bacteria (Hydrogenophaga defluvii) and hydrogenophile soil spore bacteria (Hydrogenophaga soli); the growth-promoting rhizobia is at least one of Lactobacilli mobilis (Shinella zoogloeoides), Pantoea clavuligeris (Shinella kummelianae) and Pantoea graminis (Shinella grandifolia).
4. The composite nitrogen-fixing microbial agent according to claim 1, wherein the nitrogen-fixing bacteria is Azospirillum brasiliensis (Azospirillum brasilense); the hydrogenophile is atypical hydrogenophile spore (Hydrogenophaga typica); the growth-promoting rhizobia is a zoogloeostereum species (Shinella zoogloeoides).
5. The compound nitrogen-fixing microbial agent as claimed in claim 1, wherein the Azospirillum brasiliensis (Azospirillum brasilense) is a strain with the number of CGMCC: 1.10379; the atypical hydrogenophila (Hydrogenophaga atypica) is a strain with the number of CGMCC: 1.13009; the zoogloeostereum sp (Shinella zoogloeoides) is a strain with the number of CGMCC: 1.6838.
6. The compound nitrogen-fixing microbial agent as claimed in claim 1, wherein the improved BPY culture medium takes water as a medium and comprises the following components: 3.75-6.25 g/L peptone, 1.13-1.88 g/L beef extract, 7.50-12.5 g/L sucrose, 0.12-0.19 g/L yeast extract, 1.88-3.13 g/L NaCl, 0.2g/L KH2PO4、0.8g/L K2HPO40.075-0.125 g/L magnesium sulfate heptahydrate, 0.038-0.063 g/L calcium sulfate dihydrate, 0.00005-0.0005 g/L ferric chloride, 0.00005-0.0005 g/L sodium tungstate, 0.00005-0.0005 g/L sodium molybdate and 0.00005-0.0005 g/L manganese sulfate.
7. The production method of the composite nitrogen-fixing microbial agent as claimed in claim 1, characterized by comprising the following steps:
(1) the azotobacter, the hydrogenophile and the growth-promoting rhizobia are independently cultured in corresponding culture media respectively to prepare azotobacter fermentation liquor, hydrogenophile fermentation liquor and growth-promoting rhizobia fermentation liquor: the viable count of the corresponding azotobacter, hydrogenophile and growth-promoting rhizobia in the azotobacter fermentation liquor, hydrogenophile fermentation liquor and growth-promoting rhizobia fermentation liquor is not less than 2 multiplied by 108CFU/mL；
(2) Equivalently adding the nitrogen-fixing fermentation broth, the hydrogenophile fermentation broth and the growth-promoting rhizobium fermentation broth prepared in the step (1) into an improved BPY culture medium, and fermenting for 24-48 h at 26-30 ℃ and 150-200 rpm/min to prepare a mixed fermentation broth, wherein the viable count of the three single strains in the mixed fermentation broth is not less than 1 x 106CFU/mL; the total viable count in the mixed fermentation liquor is not less than 1 multiplied by 109CFU/mL。
8. The production method of the composite nitrogen-fixing microbial agent according to claim 7,
the nitrogen-fixing bacteria culture medium takes water as a medium and comprises the following components: 0.375-0.625 g/L yeast extract, 15-25 g/L mannose, 0.2g/L monopotassium phosphate, 0.8g/L dipotassium phosphate, 0.15-0.25 g/L magnesium sulfate heptahydrate, 0.075-0.125 g/L calcium sulfate, 0.00005-0.0005 g/L ferric chloride and 0.00005-0.0005 g/L sodium molybdate;
the hydrogenophilic bacteria culture medium takes water as a medium and comprises the following components: 7.5-12.5 g/L of peptone, 2.25-3.75 g/L of beef extract and 3.75-6.25 g/L of NaCl;
the growth-promoting rhizobium culture medium takes water as a medium and comprises the following components: 7.5-12.5 g/L peptone, 2.25-3.75 g/L beef extract and 3.75-6.25 g/L NaCl.
9. The use of the composite nitrogen-fixing microbial agent of claim 1 in soybean nitrogen fixation, peanut, wheat, oat, and corn rejuvenation.
10. The use of the composite nitrogen-fixing microbial agent of claim 1 for alleviating soil acidification.
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