CN111575198A - Nitrogen-fixing microorganism composite microbial agent and application thereof - Google Patents
Nitrogen-fixing microorganism composite microbial agent and application thereof Download PDFInfo
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- CN111575198A CN111575198A CN202010306780.8A CN202010306780A CN111575198A CN 111575198 A CN111575198 A CN 111575198A CN 202010306780 A CN202010306780 A CN 202010306780A CN 111575198 A CN111575198 A CN 111575198A
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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
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/006—Waste from chemical processing of material, e.g. diestillation, roasting, cooking
- C05F5/008—Waste from biochemical processing of material, e.g. fermentation, breweries
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Abstract
The invention relates to the technical field of biological agents, in particular to a nitrogen-fixing microorganism composite agent and application thereof, wherein the composite agent comprises Bacillus licheniformis (Bacillus licheniformis) and Aspergillus fumigatus (Aspergillus fumigatus); the composite microbial inoculum can efficiently promote the accumulation of nitrogen in the preparation process of the vinasse organic fertilizer and improve the fertilizer efficiency of the vinasse organic fertilizer; and all the used strains are screened from a vinasse fermentation environment, and the utilization of the strains is friendly to ecological environment, safe and free of secondary pollution.
Description
Technical Field
The invention relates to the technical field of biological agents, in particular to a nitrogen-fixing microorganism composite agent and application thereof.
Background
The microbial fertilizer is a nontoxic, environment-friendly and environment-friendly fertilizer, and is one of important research directions for sustainable development of agriculture and forestry. It makes crops obtain nutrient supply by the life activity of specific microorganism, and has the functions of increasing crop yield, influencing the microbial structure of planting soil, improving ecological environment and the like.
The sorghum serves as a main raw material in the wine making process, and related researches find that the yield of the sorghum is increased and the grain amylopectin is accumulated when nitrogen fertilizer and phosphate fertilizer are applied, and nitrogen, phosphorus and potassium fertilizer application shortage can cause the reduction of the yield of the sorghum, particularly nitrogen fertilizer application shortage is the most obvious (Wangjinsong and the like, 2015; Sufu source and the like, 2016), so that the nitrogen content in the organic fertilizer is one of important detection indexes for applying the organic fertilizer to a sorghum-planting land.
Currently known nitrogen-fixing bacteria mainly comprise three types of self-generated nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria and combined nitrogen-fixing bacteria. The symbiotic azotobacter and the combined azotobacter are difficult to be widely applied due to strict requirements on symbiotic plants and growth environment, and the self-growing azotobacter is a main strain for manufacturing the azotobacter fertilizer. The lees, also called as distiller's grains, refers to residues produced by distilling alcohol or alcoholic beverage from grains, i.e., residues produced after brewing wine, and is commonly used as livestock feed. The vinasse is used as a main waste in the production process of the white spirit, has rich nutrient substances, can provide nutrient components required by growth and metabolism for microorganisms, is an excellent raw material for preparing organic fertilizer, can change waste into valuable, and can reduce environmental pollution. However, relatively few researches on the production of high-nitrogen-content organic fertilizer by inoculating nitrogen-fixing bacteria in vinasse are currently carried out.
Disclosure of Invention
The invention aims to provide a complex microbial inoculum and application of the complex microbial inoculum.
In one aspect, the present invention provides a complex microbial agent comprising bacillus licheniformis (bacillus licheniformis) and Aspergillus fumigatus (Aspergillus fumigatus).
In some embodiments, the Bacillus licheniformis (Bacillus licheniformis) is selected from strain MX8 with the collection number CGMCC No. 7901. The preservation date of the strain is 7 months and 8 days in 2013.
In some embodiments, the Aspergillus fumigatus (Aspergillus fumigatus) is selected from strain MM6 with the accession number cgmccno.7904. The preservation date of the strain is 7 months and 8 days in 2013.
In some embodiments, the method for culturing Bacillus licheniformis (Bacillus licheniformis) comprises: adding the strain into a liquid culture medium, culturing at 35-37 deg.C for 1-2 days to obtain a liquid seed solution, transferring to testa Tritici sterilized at 121 deg.C for 30min, and culturing at 35-37 deg.C for 8-9 days to obtain solid microbial inoculum of Bacillus licheniformis (Bacillus licheniformis) single strain.
In some embodiments, the method of culturing Aspergillus fumigatus (Aspergillus fumigatus) comprises: adding the strain into liquid culture medium, culturing at 28-30 deg.C for 2-4 days, transferring the obtained liquid seed solution onto testa Tritici sterilized at 121 deg.C for 30min, and culturing at 28-30 deg.C for 8-9 days to obtain solid microbial agent of Aspergillus fumigatus single strain.
In some embodiments, the solid microbial inoculum of the single strain of Bacillus licheniformis (Bacillus licheniformis) and Aspergillus fumigatus (Aspergillus fumigatus) in the composite microbial inoculum has a weight part ratio of (6-8) to (2-4).
On one hand, the invention also provides application of the composite microbial inoculum in preparation of nitrogen fixation products, wherein the nitrogen fixation products are selected from one or more of biological nitrogen fixation microbial inoculum, yield increasing microbial inoculum, improved fertility increasing microbial inoculum and generated nitrogen fixation enzyme microbial inoculum or fertilizer.
In some embodiments, the subject to which the nitrogen fixation product is applied is selected from the group consisting of soil, hydroponics, distillers grains.
In some embodiments, the subject is selected from one or more of a hydroponic fluid or distillers grains.
In some embodiments, the subject is selected from distillers grains.
On one hand, the invention also provides a fertilizer, which comprises the compound microbial inoculum and a fertilizer matrix; the addition amount of the compound microbial inoculum in the fertilizer is 4-10%.
In some embodiments, the amount of the complex microbial inoculum added in the fertilizer is 4% -8%.
In some embodiments, the amount of the complex microbial inoculum added in the fertilizer is 4% -6%.
In some embodiments, the complex microbial inoculum is added in an amount of 5% in the fertilizer.
On one hand, the fertilizer application method is also provided, and the method comprises the following step that when the bacterium content of the compound bacterium agent is more than or equal to 4 × 108And when CFU/g is needed, inoculating the compound microbial inoculum by the inoculation amount of more than or equal to 1 percent.
On one hand, the invention also provides application of the composite microbial inoculum in nitrogen fixation.
On one hand, the invention also provides application of the compound microbial inoculum in producing organic fertilizer by stacking vinasse.
In another aspect, the invention also provides a method for producing organic fertilizer by stacking vinasse, which comprises the following steps:
inoculating the complex microbial inoculum of claim 1 into vinasse; preferably, the bacterium content of the compound bacterium agent is more than or equal to 4 multiplied by 108 CFU/g; preferably, the inoculation amount of the inoculation is more than or equal to 1 percent.
The composite microbial inoculum can efficiently promote the accumulation of nitrogen. When the vinasse is taken as an inoculation object, the fertilizer efficiency of the vinasse organic fertilizer can be effectively improved; and all the used strains are screened from a vinasse fermentation environment, and the utilization of the strains is friendly to ecological environment, safe and free of secondary pollution.
Drawings
FIG. 1 is a graph of total nitrogen content of distillers grains from different treatments in example 3;
FIG. 2 shows the net total nitrogen increase for different treatments of distillers grains in example 3.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
EXAMPLE 1 measurement of Nitrogen-fixing ability of Strain
1.1 culture Medium formulation for experiments
Modified arbuscular mycorrhizal (Ashby) nitrogen-free liquid medium: glucose (or mannitol) 10 g; MgSO (MgSO)4.7H2O0.2g；K2HPO40.2g；K2SO40.2g；NaCl 0.2g；CaCO35g of the total weight of the mixture; 1000ml of distilled water.
1.2 detection of Nitrogen fixation Capacity of Strain
Modified nitrogen-free liquid culture medium of Asparagus officinalis (Ashby) is sterilized at 121 ℃ for 30min, then respectively inoculated with Bacillus licheniformis MX8 strain and Aspergillus fumigatus MM6 strain, the bacteria are cultured at 37 ℃, the mold is cultured at 30 ℃, after 5 days of culture, the total nitrogen content in the fermentation liquor is detected by Kjeldahl method, and the results are shown in Table 1.
TABLE 1 Total nitrogen content of fermentation broths of different strains
As can be seen from Table 1, both the Bacillus licheniformis MX8 strain and the Aspergillus fumigatus MM6 strain of the present invention have nitrogen fixation ability.
Example 2 preparation of a high-efficiency Nitrogen-fixing microbial agent
2.1 test methods
Respectively streaking and inoculating a bacillus licheniformis MX8 strain and an aspergillus fumigatus MM6 strain on beef extract peptone and YPD agar culture media, culturing at 30-37 ℃ for 24 hours, selecting single colonies, respectively inoculating the single colonies into 500mL triangular flasks containing a sterile beef extract peptone liquid culture medium and an YPD liquid culture medium, culturing at 30-37 ℃ for 48 hours at 180r/min, respectively inoculating the liquid strains of the strains on sterilized bran in an inoculation amount of 5% (v/w), fully mixing uniformly, covering with a plurality of layers of sterile gauze, culturing at room temperature, stirring for dissolving oxygen at least once every day, and culturing for 7-9 days.
2.2 test results
After 7 days of culture, the number of microorganisms can reach 4 × 108CFU/g is above; and airing the bran of the cultured bacillus licheniformis MX8 strain and aspergillus fumigatus MM6 strain, respectively and uniformly mixing according to the weight parts of 6:4, 7:3 and 8:2 to obtain the composite microbial inoculum, and preserving at normal temperature for later use.
Example 3 vinasse composting test
3.1 test materials
The complex microbial inoculum obtained in example 2; 150kg of distiller's grains per pile are piled up in 10 piles respectively.
3.2 test methods
The heap 1 is used as a blank reference (the inoculation amount is 0%), the heaps 2-4, 5-7 and 8-10 are respectively used as treatment groups for inoculating different complex microbial inoculants (different complex microbial inoculants are shown in table 2), the amounts of the inoculated complex microbial inoculants are respectively 0.5%, 1% and 1.5%, and the materials are mixed and stirred uniformly; stacking for 30 days, and detecting the total nitrogen content in the vinasse.
3.3 test results and conclusions
After 30 days of stacking, the total nitrogen content in the distiller's grains was measured, and the results are shown in Table 2 and FIG. 1.
TABLE 2 Total nitrogen content (g/kg) of distillers' grains treated differently
In the table "MX 8: MM6 "refers to the ratio of parts by weight of the bran of the Bacillus licheniformis MX8 strain and Aspergillus fumigatus MM6 strain obtained by culturing in example 2.
The net total nitrogen fixation increment for the non-treated groups after 30 days of stacking is shown in table 3 and figure 2.
TABLE 3 Total nitrogen increment for different treatments of distillers' grains
In the table "MX 8: MM6 "refers to the ratio of parts by weight of the bran of the Bacillus licheniformis MX8 strain and Aspergillus fumigatus MM6 strain obtained by culturing in example 2.
The results in Table 3 and FIG. 2 show that although inoculation with either B.licheniformis or A.fumigatus alone increased the nitrogen content in the spent grain to some extent; however, when Bacillus licheniformis and Aspergillus fumigatus are inoculated at the same time, the nitrogen fixation effect is obviously enhanced, which shows that the composite microbial inoculum of the invention has synergistic effect on nitrogen fixation.
The table shows that after 30 days of stacking, the total nitrogen detected in the 1.0% or 1.5% of the stacks was significantly higher than in the other treatments. The inoculation amount of the complex microbial inoculum is more than or equal to 1.0 percent, and the complex microbial inoculum has obvious promotion effect on nitrogen fixation of compost.
Claims (10)
1. A complex microbial inoculum, which is characterized by comprising Bacillus licheniformis (Bacillus licheniformis) and Aspergillus fumigatus (Aspergillus fumigatus).
2. The complex microbial inoculant according to claim 1, wherein the bacillus licheniformis (bacillus licheniformis) is selected from the strain MX8 with the collection number CGMCC No. 7901;
preferably, the Aspergillus fumigatus (Aspergillus fumigatus) is selected from the strain MM6 with the collection number of CGMCC NO. 7904.
3. The complex microbial inoculant according to claim 1, wherein the bacillus licheniformis (bacillus licheniformis) culture method comprises: adding the strain into a liquid culture medium, culturing at 35-37 deg.C for 1-2 days to obtain a liquid seed solution, transferring to testa Tritici sterilized at 121 deg.C for 30min, and culturing at 35-37 deg.C for 8-9 days to obtain a solid microbial inoculum of Bacillus licheniformis (Bacillus licheniformis) single strain; preferably, the method for culturing aspergillus fumigatus (aspergillus fumigatus) comprises: adding the strain into liquid culture medium, culturing at 28-30 deg.C for 2-4 days, transferring the obtained liquid seed solution onto testa Tritici sterilized at 121 deg.C for 30min, and culturing at 28-30 deg.C for 8-9 days to obtain solid microbial agent of Aspergillus fumigatus single strain;
preferably, in the composite microbial inoculum, the weight part ratio of the solid microbial inoculum of single strains of Bacillus licheniformis (Bacillus licheniformis) and Aspergillus fumigatus (Aspergillus fumigatus) is (6-8) to (2-4).
4. The application of the composite microbial inoculum of any one of claims 1 to 3 in preparing a nitrogen fixation product, wherein the nitrogen fixation product is selected from one or more of a biological nitrogen fixation microbial inoculum, a yield increasing microbial inoculum, an improved fertility increasing microbial inoculum, a produced nitrogen fixation enzyme microbial inoculum or a fertilizer.
5. The use of claim 4, wherein the nitrogen fixation product is applied to a subject selected from one or more of soil, hydroponics, distillers' grains;
preferably, the subject is selected from one or more of water culture solution or vinasse;
preferably, the subject is selected from distillers grains.
6. A fertilizer, which comprises the complex microbial agent of any one of claims 1 to 3 and a fertilizer substrate; the addition amount of the compound microbial inoculum in the fertilizer is 4-10 percent;
preferably, the addition amount of the compound microbial inoculum in the fertilizer is 4% -8%;
preferably, the addition amount of the compound microbial inoculum in the fertilizer is 4% -6%;
preferably, the addition amount of the compound microbial inoculum in the fertilizer is 5%.
7. A fertilizing method is characterized by comprising the following steps that when the bacterium content of the compound bacterium agent as claimed in any one of claims 1-3 is more than or equal to 4 × 108And when CFU/g is needed, inoculating the compound microbial inoculum by the inoculation amount of more than or equal to 1 percent.
8. The use of the complex microbial inoculum of any one of claims 1 to 3 in nitrogen fixation.
9. The use of the complex microbial inoculum of any one of claims 1 to 3 in the production of organic fertilizer by vinasse accumulation.
10. A method for producing organic fertilizer by stacking vinasse is characterized by comprising the following steps:
inoculating the complex microbial inoculum according to any one of claims 1 to 3 into vinasse;
preferably, the bacterium content of the compound bacterium agent is more than or equal to 4 × 108CFU/g；
Preferably, the inoculation amount of the inoculation is more than or equal to 1 percent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113979790A (en) * | 2021-08-26 | 2022-01-28 | 中国烟草总公司贵州省公司 | Coal gangue microbial fungus fertilizer and preparation method thereof |
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| CN103898032A (en) * | 2014-04-25 | 2014-07-02 | 江苏联海生物科技有限公司 | Complex microbial inoculant and method for preparing organic fertilizer by using same |
| CN104059862A (en) * | 2014-04-02 | 2014-09-24 | 贵州茅台酒股份有限公司 | Cellulose degradation bacteria agent and raw material strain, and preparation methods and application of cellulose degradation bacteria agent and raw material strain |
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| CN104059862A (en) * | 2014-04-02 | 2014-09-24 | 贵州茅台酒股份有限公司 | Cellulose degradation bacteria agent and raw material strain, and preparation methods and application of cellulose degradation bacteria agent and raw material strain |
| CN103898032A (en) * | 2014-04-25 | 2014-07-02 | 江苏联海生物科技有限公司 | Complex microbial inoculant and method for preparing organic fertilizer by using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113979790A (en) * | 2021-08-26 | 2022-01-28 | 中国烟草总公司贵州省公司 | Coal gangue microbial fungus fertilizer and preparation method thereof |
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