CN114774325A - Compound microbial agent for promoting utilization of crop nitrogen, preparation method and application - Google Patents

Compound microbial agent for promoting utilization of crop nitrogen, preparation method and application Download PDF

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CN114774325A
CN114774325A CN202210509385.9A CN202210509385A CN114774325A CN 114774325 A CN114774325 A CN 114774325A CN 202210509385 A CN202210509385 A CN 202210509385A CN 114774325 A CN114774325 A CN 114774325A
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fermentation
fermentation product
culture medium
bacteria
activated
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户正荣
尹华群
蔡海林
方明
曹明锋
刘天波
孔午圆
刘勇军
靳志丽
周志成
黎亮志
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TOBACCO AGRICULTURAL EXPERIMENT STATION OF CENTRAL-SOUTH CHINA
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TOBACCO AGRICULTURAL EXPERIMENT STATION OF CENTRAL-SOUTH CHINA
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    • 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
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/32—Yeast
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/34—Aspergillus
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/38—Trichoderma
    • 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
    • C12N1/14—Fungi; 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor

Abstract

The invention discloses a compound microbial agent for promoting the utilization of crop nitrogen, which comprises the following components in percentage by mass: 20-30% of a nitrosospira fermentation product, 20-30% of a lactobacillus fermentation product, 20-30% of a yeast fermentation product, 10-20% of an aspergillus niger fermentation product and 10-20% of a trichoderma reesei fermentation product, wherein each fermentation product comprises a corresponding bacterium and a metabolite produced by the corresponding bacterium. The compound microbial agent contains various microbes including bacteria, fungi and the like, metabolites thereof and enzymes. Each strain has respective emphasis and can complement advantages, so that the strain has the advantages of various functions and metabolites, no antagonism, difficult inhibition and the like; and can promote the conversion of ammonium nitrogen into nitrite nitrogen, activate insoluble phosphorus and calcium in the soil to change the insoluble phosphorus and calcium into an effective state, improve the nitrogen absorption and nitrogen metabolism level of a host, and promote the growth of crops and the absorption of nutrient substances.

Description

Compound microbial agent for promoting utilization of crop nitrogen, preparation method and application
Technical Field
The invention relates to the technical field of microbial agents, in particular to a compound microbial agent for promoting utilization of crop nitrogen, a preparation method and application thereof.
Background
In the crop cultivation process, nitrogen is the most important element influencing growth, development, yield and quality. At present, the requirement of nitrogen in the growth process of crops is met by applying a large amount of nitrogen fertilizer in the production process. However, the utilization efficiency of nitrogen fertilizer by crops is only 10% -15%, and the rest large amount of nitrogen is lost with underground water or converted into nitrogen to return to the atmosphere.
Nitrogen reuse is particularly important for the development of plant neogenetic tissues, and more than 50% of nitrogen is derived from nitrogen reuse in the reproductive growth phase. Improving the nitrogen utilization efficiency of crops to ensure the crop production and environmental safety under the low nitrogen fertilizer input is an important subject in the future sustainable agricultural development.
Microorganisms participate in the nitrogen cycle process through nitrification and denitrification in the soil. At present, the domestic compound microbial inoculant has single selected strain and low nitrogen conversion efficiency.
Disclosure of Invention
Therefore, it is necessary to provide a composite microbial inoculum for promoting the utilization of nitrogen in crops, a preparation method and an application thereof aiming at the problems of single strain, lack of diversity of functions and low conversion efficiency of the traditional composite microbial inoculum.
In a first aspect of the application, a composite microbial agent for promoting nitrogen utilization of crops is provided. The compound microbial agent for promoting the utilization of the crop nitrogen comprises the following components in percentage by mass: 20-30% of a nitrosospira fermentation product, 20-30% of a lactobacillus fermentation product, 20-30% of a yeast fermentation product, 10-20% of an aspergillus niger fermentation product and 10-20% of a trichoderma reesei fermentation product, wherein each fermentation product comprises corresponding bacteria and metabolites produced by the corresponding bacteria, and each metabolite comprises enzymes.
Optionally, the compound microbial agent for promoting the utilization of crop nitrogen comprises the following components in percentage by mass: 20% of nitrosospira fermentation product, 30% of lactobacillus fermentation product, 30% of yeast fermentation product, 10% of aspergillus niger fermentation product and 10% of trichoderma reesei fermentation product.
In a second aspect of the present application, a preparation method of the complex microbial inoculant is provided, which comprises the following steps:
respectively inoculating the activated seeds of the bacteria and the activated seeds of the fungi into corresponding fermentation tanks for fermentation; the bacteria comprise nitrosospira and lactobacillus, and the fungi comprise yeast, aspergillus niger and trichoderma reesei;
mixing the fermentation liquors according to the weight percentage of each fermentation product in claim 1 or 2 to obtain the compound microbial agent; or inoculating the activated seeds of all bacteria into a fermentation tank at the same time for fermentation, then inoculating the activated seeds of all fungi into the same fermentation tank at the same time for co-fermentation, and taking the fermentation liquor as the compound microbial agent; the bacteria include Nitrospira and Lactobacillus, and the fungi include yeast, Aspergillus niger and Trichoderma reesei.
Optionally, the bacterial seeds and the fungal seeds are activated in a first culture medium for 1-2 times, respectively, and the first culture medium includes: 0.3g/L glucose, 0.2g/L yeast extract powder, KH2PO40.01g/L，MgSO40.01g/L, and the pH of the first culture medium is 5.5-6.0.
Optionally, the step of inoculating the activated seeds of each bacterium and the activated seeds of each fungus into corresponding fermenters, respectively, to perform fermentation includes:
inoculating the activated seeds of the bacteria and the activated seeds of the fungi into corresponding fermenters respectively at an inoculation concentration of 10% (V/V), and fermenting in a first culture medium at 35 ℃ and a dissolved oxygen concentration of 60-100%, wherein the inoculation amount is 10% (V/V), and the first culture medium comprises: glucose 0.3g/L, yeast extract powder 0.2g/L, KH2PO40.01g/L，MgSO40.01g/L, the pH of the first culture medium is 5.5-6.0;
after fermenting for 24 hours, supplementing a glucose solution into each fermentation tank, keeping the concentration of glucose in the first culture medium at 5-10g/L, and continuing to ferment;
when the total fermentation time reaches 72h and the total fermentation amount is 3/5 corresponding to the volume of the fermentation tank, stopping fermentation to obtain fermentation liquor of corresponding bacteria.
Optionally, the step of mixing the fermentation broths further comprises:
diluting each fermentation broth with water to a final cell density of 1 × 108cfu/ml。
Optionally, after the activated seeds of the respective bacteria are simultaneously inoculated into a fermentation tank for fermentation, the activated seeds of the respective fungi are simultaneously inoculated into the same fermentation tank for co-fermentation, and the step of co-fermentation comprises:
inoculating the seeds of the activated bacteria into a fermentation tank at an inoculation concentration of 10% (V/V), and fermenting in a first culture medium at 35 ℃ and a dissolved oxygen concentration of 60-100%, wherein the first culture medium comprises: 0.3g/L glucose, 0.2g/L yeast extract powder, KH2PO40.01g/L，MgSO40.01g/L, the pH of the first culture medium is 5.5-6.0;
after fermenting for 24h, supplementing glucose solution in the fermentation tank, keeping the glucose concentration in the first culture medium at 5-10g/L, inoculating the activated seeds of each fungus at an inoculation concentration of 0.5% (V/V) and continuing fermentation;
when the total fermentation time reaches 72h and the total fermentation amount is 3/5 of the volume of the fermentation tank, stopping fermentation to obtain fermentation liquor.
Optionally, in each activated bacterium, the inoculation ratio of the nitrosospira to the lactobacillus is 1-3: 5-7;
in the activated fungi, the inoculation ratio of the saccharomycetes, the aspergillus niger and the trichoderma reesei is 1-3: 1-5: 1 to 5.
Optionally, the aeration rate is adjusted to 1.0vvm (gas volume/liquid volume/min), and the stirring rate is set to 50-150 rpm, so that the dissolved oxygen concentration is 60-100%.
In a third aspect of the present application, an application of the complex microbial inoculant or the complex microbial inoculant prepared by the preparation method in crop cultivation is provided.
The compound microbial agent contains various microorganisms including bacteria, fungi and the like, metabolites thereof and enzymes. Nitrosospira and lactobacillus are bacteria, and yeast, aspergillus niger and trichoderma reesei are fungi. The bacteria can utilize the glucose generated after the fungi decompose the cellulose to grow, and simultaneously promote the nitrogen transformation and generate the plant active substances to promote the plant growth, thereby having the advantage of low required nutrient conditions. Each strain has respective emphasis and can complement advantages, so that the strain has the advantages of various functions and metabolites, no antagonism, difficult inhibition and the like; and can promote the ammonium nitrogen to be converted into nitrite nitrogen, activate the indissolvable phosphorus and calcium in the soil to be changed into an effective state, improve the nitrogen absorption and nitrogen metabolism level of a host, and promote the growth of crops and the absorption of nutrient substances.
Drawings
Fig. 1 is a flowchart of a method for preparing a complex microbial inoculant according to an embodiment of the present disclosure.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The application provides a compound microbial agent for promoting the utilization of crop nitrogen, which comprises the following components in percentage by mass: 20-30% of a nitrosospira fermentation product, 20-30% of a lactobacillus fermentation product, 20-30% of a yeast fermentation product, 10-20% of an aspergillus niger fermentation product and 10-20% of a trichoderma reesei fermentation product, wherein each fermentation product comprises corresponding bacteria and metabolites produced by the corresponding bacteria, and each metabolite comprises enzymes.
In the aspect of raw material selection: lactobacillus is a common species of bacteria, widely distributed in grains, plant products and soil all over the world, is an important fermentation industrial strain, can decompose ammonia gas, and produce amylase, acid protease, cellulase, pectinase, glucose oxidase, citric acid, gluconic acid and the like.
The yeast, the trichoderma reesei and the aspergillus niger do not produce toxin while producing enzyme in the production process, can produce various metabolites, accelerate nitrogen conversion, activate soil and promote crop production. Nitrosospirillum is ammonia oxidizing bacteria in soil, is a chemoautotrophic microorganism, has no specificity to plants, utilizes ammonia as a substrate, oxidizes the ammonia into nitrite under the action of ammonia oxidase and hydroxylamine oxidoreductase, can generate plant active substances and promotes the growth of crops.
In the aspect of strain collocation: because microorganisms in soil are relatively complex, and a single bacterium is easily antagonized by some microorganisms in soil, the effect of the microbial inoculum is not ideal, so that the aspergillus niger, trichoderma reesei, saccharomycetes and ammonia oxidizing microorganisms are reasonably matched according to the growth characteristics and functional characteristics of different microorganisms, no antagonism exists among strains, the aspergillus niger, the trichoderma reesei and the saccharomycetes belong to fungi, and all the aspergillus niger, the trichoderma reesei and the saccharomycetes can generate cellulase, protease, amylase, small molecular organic acid and the like, and the inhibition probability is reduced. The aspergillus niger has strong acid production capacity, has the functions of dissolving phosphorus and calcium, and can better dissolve mineral nutrients in soil for crops and other microorganisms. The cellulase producing capability of the trichoderma reesei is obviously superior to that of other fungi, and can provide a good carbon source for other microorganisms. The bacteria can utilize the fungi to decompose the cellulose to generate glucose for growth, and simultaneously promote the nitrogen transformation and generate plant active substances for promoting the plant growth. Each strain has its own emphasis point, so that the advantages can be complemented. In addition, the compound microbial agent of the invention comprises bacteria, various enzymes and other metabolites (such as organic acid) produced by the bacteria, and metabolites such as the enzymes can play a role immediately after entering soil, so that organic macromolecules such as cellulose in the soil are decomposed into small molecules, and the growth of the bacteria is promoted.
Compared with the prior art, the invention has the following beneficial effects: (1) the compound microbial agent provided by the invention contains various microorganisms including bacteria, fungi and the like, metabolites thereof and enzymes thereof, and has the advantages of low required nutritional conditions, various functions and metabolites, no antagonism, difficult inhibition and the like. And can promote the ammonium nitrogen to be converted into nitrite nitrogen, activate the indissolvable phosphorus and calcium in the soil to be changed into an effective state, improve the nitrogen absorption and nitrogen metabolism level of a host, and promote the growth of crops and the absorption of nutrient substances. (2) The invention can effectively improve the quantity and variety of beneficial microbial communities in soil, can form a protective layer around the rhizosphere of plants, and prevents pathogenic microorganisms from invading the plants. (4) The active substances generated by the composite microbial agent provided by the invention can promote the growth of crops and achieve the effects of increasing yield and improving quality.
Optionally, the composition comprises the following components in percentage by mass: 20% of nitrosospira fermentation product, 30% of lactobacillus fermentation product, 30% of yeast fermentation product, 10% of aspergillus niger fermentation product and 10% of trichoderma reesei fermentation product. . Under the condition, the nitrogen absorption and nitrogen metabolism level of the host can be further improved, and the growth of crops and the absorption of nutrient substances are promoted.
In a second aspect of the present application, a method for preparing a complex microbial inoculant is provided. The compound microbial agent can be prepared by respectively fermenting bacteria and fungi and then mixing fermentation liquids. In some of these embodiments, referring to fig. 1, the method of making comprises the steps of:
s100: inoculating the activated seeds of the bacteria and the activated seeds of the fungi into corresponding fermentation tanks respectively at an inoculation concentration of 10% (V/V) for fermentation; the bacteria include Nitrospira and Lactobacillus, and the fungi include yeast, Aspergillus niger and Trichoderma reesei.
S200: and mixing the fermentation liquids according to the weight percentage of the fermentation products to obtain the compound microbial agent.
Under the condition, nitrosospira, lactobacillus, saccharomycetes, aspergillus niger and trichoderma reesei are respectively cultured to obtain corresponding fermentation broth, and then the fermentation broths are mixed according to mass percentage to obtain the compound microbial agent.
In the above embodiments, the complex microbial inoculum is prepared by a separate culture method, or can be prepared by a system of co-culture of bacteria and fungi, and in other embodiments, the preparation method comprises the following steps:
inoculating the activated seeds of all bacteria into a fermentation tank at the same time for fermentation, then inoculating the activated seeds of all fungi into the same fermentation tank at the same time for co-fermentation, and taking the fermentation liquor as the compound microbial agent; the bacteria include nitrosospira and lactobacillus, and the fungi include yeast, aspergillus niger and trichoderma reesei.
Optionally, the bacterial seed and the fungal seed are activated 1-2 times in a first culture medium, respectively, the first culture medium comprising: 0.3g/L glucose, 0.2g/L yeast extract powder, KH2PO40.01g/L，MgSO40.01g/L, the pH of the first medium is 5.5-6.0.
The activation may be carried out by inoculating each species of bacteria and fungi into the first medium separately and culturing in a shaker (120rpm,35 ℃). The culture medium can make the activity of bacteria or fungi strong, and after the culture medium is transplanted and fermented, the bacteria or fungi can quickly grow, thereby shortening the fermentation time and ensuring the production level.
Optionally, the step of inoculating the activated seeds of each bacterium and the activated seeds of each fungus into corresponding fermenters, respectively, to perform fermentation includes:
respectively inoculating the activated seeds of the bacteria and the activated seeds of the fungi into corresponding fermentation tanks, and fermenting in a first culture medium under the conditions of 35 ℃ and dissolved oxygen concentration of 60-100%, wherein the inoculation amount is 10% (V/V), and the first culture medium comprises: glucose 0.3g/L, yeast extract powder 0.2g/L, KH2PO40.01g/L，MgSO40.01g/L, the pH of the first culture medium is 5.5-6.0;
after fermenting for 24 hours, supplementing a glucose solution into each fermentation tank, keeping the concentration of glucose in the first culture medium at 5-10g/L, and continuing to ferment;
when the total fermentation time reaches 72h and the total fermentation amount is 3L, stopping fermentation to obtain fermentation liquor of corresponding bacteria.
In this production method, the activated seeds of each bacterium and the activated seeds of each fungus are inoculated into respective fermentors and pure-cultured. For example, the mixture is inoculated into a 5L fermentation tank for fermentation, the inoculation amount is 10% (V/V), the total fermentation time is 72h, and the total fermentation amount is 3L. Keeping the Dissolved Oxygen (DO) concentration at about 60-100% at 35 ℃, fermenting for 24h, feeding a high-concentration sterile glucose solution (50%) into a fermentation tank through a peristaltic pump for feeding, maintaining the glucose concentration in a culture medium at 5-10g/L, and not adjusting the pH value of the fermentation liquid in the fermentation process. Specifically, pH and DO can be measured by sensors on the fermentor; the pH, cell density and DO values were recorded every 6h and the cell density was calculated microscopically by means of a haemocytometer. After staining the microbial cells with carbolic acid red dye for 2 minutes, they were observed under a microscope 1000X.
Optionally, the step of mixing each fermentation broth further comprises:
diluting each fermentation broth with water to final cell density of 1 × 108cfu/ml。
The fermentation product can be diluted with sterile deionized water to a final cell density of 1X 108cfu/ml (colony forming units/ml).
Optionally, the step of simultaneously inoculating the activated seeds of the bacteria into the fermentation tank for fermentation, and then simultaneously inoculating the activated seeds of the fungi into the fermentation tank for co-fermentation comprises:
inoculating the seeds of the activated bacteria into a fermentation tank at an inoculation concentration of 10% (V/V), and fermenting in a first culture medium at 35 ℃ and a dissolved oxygen concentration of 60-100%, wherein the first culture medium comprises: 0.3g/L glucose, 0.2g/L yeast extract powder, KH2PO40.01g/L MgSO40.01g/L, the pH of the first culture medium is 5.5-6.0;
after 24h of fermentation, the fermentation tank is supplemented with glucose solution, the glucose concentration in the first culture medium is kept at 5-10g/L, and the activated seeds of each fungus are inoculated at an inoculation concentration of 0.5% (V/V) for continuous fermentation;
and when the total fermentation time reaches 72 hours and the total fermentation amount is 3/5 corresponding to the volume of the fermentation tank, stopping fermentation to obtain fermentation liquor. For example, when the volume of the fermentation tank is 5L and the total fermentation amount is 3L, the fermentation is stopped.
In the preparation method, the activated seeds of each bacterium and the activated seeds of each fungus are inoculated in sequence into the same fermentation tank for co-culture. All the bacterial seed liquid is inoculated into a fermentation tank for fermentation for 24 hours, and then the fungal seed liquid is inoculated into a bacterial fermentation system according to the inoculation concentration of 0.5% (v/v).
Optionally, in each activated bacterium, the inoculation ratio of the nitrosospira to the lactobacillus is 1-5: 1-5;
in the activated fungi, the inoculation ratio of the yeast and one of aspergillus niger and trichoderma reesei is 1-5: 1 to 5.
Optionally, the aeration rate is adjusted to 1.0vvm (gas volume/liquid volume/min), and the stirring rate is set to 50-150 rpm, so that the dissolved oxygen concentration is 60-100%.
In a third aspect of the application, an application of the compound microbial agent or the compound microbial agent prepared by the preparation method in crop cultivation is provided. For example, the method can be applied to cultivation of flue-cured tobacco.
The compound microbial agent contains various microorganisms including bacteria, fungi and the like, metabolites thereof and enzymes. Nitrosospira and lactobacillus are bacteria, and yeast, aspergillus niger and trichoderma reesei are fungi. The bacteria can utilize the glucose generated after the fungi decompose the cellulose to grow, and simultaneously promote the nitrogen transformation and generate the plant active substances to promote the plant growth, thereby having the advantage of low required nutrient conditions. Each strain has respective emphasis and can complement advantages, so that the strain has the advantages of various functions and metabolites, no antagonism, difficult inhibition and the like; and can promote the conversion of ammonium nitrogen into nitrite nitrogen, activate insoluble phosphorus and calcium in the soil to change the insoluble phosphorus and calcium into an effective state, improve the nitrogen absorption and nitrogen metabolism level of a host, and promote the growth of crops and the absorption of nutrient substances.
The technical solution of the present application is described below with reference to specific embodiments.
Example 1
The nitrosation spirillum, lactobacillus, saccharomycete, aspergillus niger and trichoderma reesei are respectively inoculated into a first culture medium and are placed in a shaking table (120rpm,35 ℃) for culture, thereby carrying out activation. The first medium comprises: glucose 0.3g/L, yeast extract powder 0.2g/L, KH2PO40.01g/L，MgSO40.01g/L, and the pH of the first culture medium is 5.5-6.0.
The activated seeds of the bacteria and the activated seeds of the fungi are respectively inoculated into corresponding fermenters to be purely cultured in a first culture medium, and the inoculation amount is 10 percent (V/V). The aeration rate is adjusted to 1.0vvm (gas volume/liquid volume/min) at 35 ℃, the stirring rate is 50-150 rpm, and the Dissolved Oxygen (DO) concentration is maintained at about 60-100%, so as to perform fermentation.
After fermenting for 24h, feeding high-concentration sterile glucose solution (50%) into a fermentation tank by a peristaltic pump for feeding, maintaining the glucose concentration in the culture medium at 5-10g/L, and continuing fermenting;
when the total fermentation time reaches 72h and the total fermentation amount is 3L, stopping fermentation to obtain fermentation liquor of corresponding bacteria.
Diluting the fermentation product of each fermentation liquid with sterile deionized water to final cell density of 1 × 108cfu/ml. And mixing to obtain the compound microbial agent for promoting the utilization of the nitrogen of the crops. The compound microbial agent comprises the following components in percentage by mass: 20% of nitrosospira fermentation product, 30% of lactobacillus fermentation product, 30% of yeast fermentation product, 10% of aspergillus niger fermentation product and 10% of trichoderma reesei fermentation product.
Example 2
The difference from the embodiment 1 is that the compound microbial agent comprises the following components in percentage by mass: 30% of nitrosospira fermentation product, 20% of lactobacillus fermentation product, 20% of yeast fermentation product, 20% of aspergillus fermentation product and 20% of trichoderma reesei fermentation product. The rest is the same as example 1.
Example 3
The difference from the example 1 is that the compound microbial agent comprises the following components in percentage by mass: 15% of nitrosospira fermentation product, 25% of lactobacillus fermentation product, 25% of yeast fermentation product, 15% of aspergillus niger fermentation product and 20% of trichoderma reesei fermentation product. The rest is the same as example 1.
Example 4
The nitrosation spirillum, lactobacillus, saccharomycete, aspergillus niger and trichoderma reesei are respectively inoculated into a first culture medium and are placed in a shaking table (120rpm,35 ℃) for culture, thereby carrying out activation. The first medium comprises: 0.3g/L glucose, 0.2g/L yeast extract powder, KH2PO40.01g/L，MgSO40.01g/L, the pH of the first medium is 5.5-6.0.
The activated seeds of each bacterium and the activated seeds of each fungus are respectively inoculated into corresponding fermentors to be purely cultured in a first culture medium, and the inoculation amount is 10% (V/V). The fermentation is carried out at 35 ℃ with the aeration rate adjusted to 1.0vvm (gas volume/liquid volume/min) and the stirring rate 50-150 rpm, so as to maintain the Dissolved Oxygen (DO) concentration at about 60-100%.
After fermenting for 24h, high-concentration sterile glucose solution (50%) is fed into a fermentation tank by a peristaltic pump for feeding, the glucose concentration in the culture medium is maintained at 5-10g/L, and the activated seeds of each fungus are inoculated at the inoculation concentration of 0.5% (V/V) for continuous fermentation. In the activated fungi, the inoculation ratio of the yeast, aspergillus niger and trichoderma reesei is 1-3: 1-5: 1 to 5.
When the total fermentation time reaches 72h and the total fermentation amount is 3/5 of the volume of the fermentation tank, stopping fermentation to obtain fermentation liquor. Diluting the fermentation product of the fermentation liquid with sterile deionized water to a final cell density of 1 × 108cfu/ml, and obtaining the compound microbial agent for promoting the utilization of the nitrogen of the crops. The measured compound microbial agent comprises the following components in percentage by mass: 20% of nitrosospira fermentation product, 30% of lactobacillus fermentation product, 30% of yeast fermentation product, 10% of aspergillus niger fermentation product and 10% of trichoderma reesei fermentation product.
Application examples
The test site is a test base of a hoeing garden of the Hunan agricultural university, and the test variety is tobacco K326. The soil type tested was rice soil. The test was conducted at 3-7 months 2021. The transplanting time of the tobacco plants is 2021 year, 3 months and 28 days. The experiment designed 2 treatments (with and without the complex microbial inoculum of example 1) in total, 30 pots each. The caliber of the pot mouth is 35cm, the height is 35cm, and each pot is filled with 10kg of soil. The application mode of the microbial functional bacteria agent is root irrigation. The application period is root extension period. And ensuring that other agronomic measures are the same between the treatment of applying the microbial inoculum and the treatment of not applying the microbial inoculum and are carried out according to the local high-quality tobacco leaf production technical specification. In the treatment of applying the microbial inoculum, 20mL of microbial inoculum of each plant of the tobacco plants is diluted by 10 times and then irrigated to roots.
Measurement items: and (4) selecting representative tobacco plants 80 days after the microbial inoculum is applied, and determining 6 tobacco agronomic traits comprising plant height, stem circumference, effective leaf number, maximum leaf length, maximum leaf width and the like. Destructive sampling, determining fresh weight, activity and volume of the root system of the tobacco plant, and determining the dry matter quantity of the root system after drying, wherein the dry matter quantity comprises nitrate nitrogen, ammonium nitrogen, total nitrogen and organic nitrogen content in the sample, and the concentration comprises amino acid, chlorophyll and nicotine. The method comprises the steps of shaking off blocky soil after a whole tobacco plant is pulled out, collecting rhizosphere soil by using a fine brush, dividing the soil into two parts, using one part of the soil for molecular biology experiments, using the other part of the soil for measuring soil physicochemical properties including concentrations of pH, organic matters, soil soluble carbon, organic carbon, total carbon, nitrate nitrogen, ammonium nitrogen and total nitrogen, and researching influences of spraying bactericides with different concentrations in different application periods on soil nitrogen efficient transformation and tobacco nitrogen efficient utilization.
The experimental results are as follows: after the microbial inoculum is applied, the experimental group applying the microbial inoculum is obviously superior to the control group not applying the microbial inoculum in various agronomic characters of tobacco: after the microbial inoculum is applied, the root weight is increased to 12.13 +/-4.03 g from 11.29 +/-1.62 g of the control; the stem weight is increased from 36.63 +/-5.11 g of the control to 43.80 +/-12.54 g; the leaf weight is increased from 95.35 +/-8.91 g of the control to 97.92 +/-11.88 g; the leaf number is increased to 17.8 +/-2.77 from 16.40 +/-1.14 of the control; the leaf length is increased from 62.29 plus or minus 2.82cm of the control to 63.25 plus or minus 3.53 cm; the leaf width is increased from 17.71 +/-2.11 cm of the control to 18.55 +/-1.66 cm; the plant height is increased to 113.42 +/-14.02 cm from 109.00 +/-9.12 cm of a control; the stem circumference increased from 5.68 + -0.13 cm of the control to 6.04 + -0.30 cm. The pH value of the soil has no obvious change, the content of organic matters in the soil is increased to 26.43 +/-0.94 g/kg from 24.11 +/-0.61 g/kg of a contrast, the content of total nitrogen is increased to 1.41 +/-0.09 g/kg from 1.34 +/-0.05 g/kg of the contrast, the content of total phosphorus is increased to 0.56 +/-0.03 g/kg from 0.50 +/-0.05 g/kg of the contrast, the content of total potassium is increased to 14.93 +/-0.61 g/kg from 14.00 +/-0.55 g/kg of the contrast, the content of quick-acting nitrogen is increased to 92.30 +/-6.22 mg/kg from 89.72 +/-4.22 mg/kg of the contrast, the content of quick-acting phosphorus is increased to 50.43 +/-4.26 mg/kg from 45.75 +/-7.04 mg/kg of the contrast, and the content of quick-acting potassium is increased to 48.35 +/-2.33 mg/kg from 44.35 +/-7.04 mg/kg of the contrast.
The compound microbial agent has the advantages of promoting nitrogen conversion, generating plant active substances and promoting plant growth, has low required nutritional conditions, can promote ammonium nitrogen to be converted into nitrite nitrogen, activate insoluble phosphorus and calcium in soil to be changed into an effective state, improve the nitrogen absorption and nitrogen metabolism level of a host, and promote the growth of crops and the absorption of nutrients.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims (10)

1. The compound microbial agent for promoting the utilization of crop nitrogen is characterized by comprising the following components in percentage by mass: 20-30% of a nitrosospira fermentation product, 20-30% of a lactobacillus fermentation product, 20-30% of a yeast fermentation product, 10-20% of an aspergillus niger fermentation product and 10-20% of a trichoderma reesei fermentation product, wherein each fermentation product comprises a corresponding bacterium and a corresponding metabolite produced by the bacterium, and each metabolite comprises an enzyme.
2. The compound microbial agent according to claim 1, which comprises the following components in percentage by mass: 20% of nitrosospira fermentation product, 30% of lactobacillus fermentation product, 30% of yeast fermentation product, 10% of aspergillus niger fermentation product and 10% of trichoderma reesei fermentation product.
3. A method for preparing the complex microbial inoculant of claim 1 or 2, comprising the following steps:
respectively inoculating the activated seeds of the bacteria and the activated seeds of the fungi into corresponding fermentation tanks for fermentation; the bacteria comprise nitrosospira and lactobacillus, and the fungi comprise yeast, aspergillus niger and trichoderma reesei;
mixing the fermentation liquors according to the weight percentage of each fermentation product in claim 1 or 2 to obtain the compound microbial agent; or inoculating the activated seeds of all bacteria into a fermentation tank at the same time for fermentation, then inoculating the activated seeds of all fungi into the same fermentation tank at the same time for co-fermentation, and taking the fermentation liquor as the compound microbial agent; the bacteria include Nitrospira and Lactobacillus, and the fungi include yeast, Aspergillus niger and Trichoderma reesei.
4. The method for preparing a complex microbial inoculant according to claim 3, wherein the seeds of said bacteria and said fungi are activated 1-2 times in a first culture medium respectively, said first culture medium comprising: glucose 0.3g/L, yeast extract powder 0.2g/L, KH2PO40.01g/L，MgSO40.01g/L, and the pH of the first culture medium is 5.5-6.0.
5. The method for preparing a complex microbial inoculant according to claim 3, wherein the step of inoculating the activated seeds of each bacterium and the activated seeds of each fungus into corresponding fermenters, and fermenting comprises:
inoculating the activated seeds of the bacteria and the activated seeds of the fungi into corresponding fermenters respectively at an inoculation concentration of 10% (V/V), and fermenting in a first culture medium at 35 ℃ and a dissolved oxygen concentration of 60-100%, wherein the inoculation amount is 10% (V/V), and the first culture medium comprises: glucose 0.3g/L, yeast extract powder 0.2g/L, KH2PO40.01g/L，MgSO40.01g/L, the pH of the first culture medium is 5.5-6.0;
after fermenting for 24 hours, supplementing a glucose solution into each fermentation tank, keeping the concentration of glucose in the first culture medium at 5-10g/L, and continuing to ferment;
and when the total fermentation time reaches 72h and the total fermentation amount is 3/5 of the volume of the corresponding fermentation tank, stopping fermentation to obtain fermentation liquor of the corresponding bacteria.
6. The method for preparing a complex microbial inoculant according to claim 3, wherein the step of mixing the fermentation liquors further comprises:
diluting each fermentation broth with water to a final cell density of 1 × 108cfu/ml。
7. The method for preparing a complex microbial inoculant according to claim 3, wherein the step of simultaneously inoculating the activated seeds of each bacterium into a fermentation tank for fermentation and then inoculating the activated seeds of each fungus into the same fermentation tank for co-fermentation comprises:
inoculating the activated seeds of each bacterium into a fermentation tank at an inoculation concentration of 10% (V/V), and fermenting in a first culture medium at 35 ℃ and a dissolved oxygen concentration of 60-100%, wherein the first culture medium comprises: 0.3g/L glucose, 0.2g/L yeast extract powder, KH2PO40.01g/L，MgSO40.01g/L, the pH of the first culture medium is 5.5-6.0;
after fermenting for 24h, supplementing glucose solution in the fermentation tank, keeping the glucose concentration in the first culture medium at 5-10g/L, inoculating the activated seeds of each fungus at an inoculation concentration of 0.5% (V/V) and continuing fermentation;
when the total fermentation time reaches 72h and the total fermentation amount is 3/5 of the volume of the fermentation tank, stopping fermentation to obtain the fermentation liquid.
8. The method for preparing a complex microbial inoculant according to claim 3, wherein the activated bacteria have an inoculation ratio of said Nitrospira and said Lactobacillus of 1-3: 5-7;
in the activated fungi, the inoculation ratio of the saccharomycetes, the aspergillus niger and the trichoderma reesei is 1-3: 1-5: 1 to 5.
9. The method for preparing a complex microbial inoculant according to claim 5 or 7, wherein the aeration rate is adjusted to 1.0vvm (gas volume/liquid volume/min) and the stirring rate is set to 50-150 rpm, such that the dissolved oxygen concentration is 60-100%.
10. The application of the compound microbial inoculant of claim 1 or 2 or the compound microbial inoculant prepared by the preparation method of any one of claims 3 to 9 in crop cultivation.
CN202210509385.9A 2022-05-11 2022-05-11 Compound microbial agent for promoting utilization of crop nitrogen, preparation method and application Pending CN114774325A (en)

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