CN112458017A - Bacillus subtilis XDK06 and application thereof - Google Patents
Bacillus subtilis XDK06 and application thereof Download PDFInfo
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- CN112458017A CN112458017A CN202011386084.9A CN202011386084A CN112458017A CN 112458017 A CN112458017 A CN 112458017A CN 202011386084 A CN202011386084 A CN 202011386084A CN 112458017 A CN112458017 A CN 112458017A
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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
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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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
- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
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- 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
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention discloses a bacillus subtilis XDK06 which is preserved in Guangdong province microorganism strain preservation center in 10 and 30 months in 2020, wherein the preservation number is GDMCC No: 61260 of a solvent; the invention also discloses a culture method of the bacillus subtilis XDK06 and application of the bacillus subtilis XDK06 in organic fertilizer fermentation. The bacillus subtilis XDK06 has anaerobic fermentation property, can ferment decomposed organic materials under anaerobic conditions, does not need aeration, can keep the fermentation temperature at a medium temperature of 30-50 ℃, is environment-friendly, safe and energy-saving in the fermentation process, only needs to turn the pile for 1-2 times in the whole fermentation process, greatly reduces the labor intensity, can fully decompose the organic materials, retains the nutrient components in the organic materials to a greater extent, produces the fertilizer with buoyancy, cannot immediately sink to the water bottom after being applied to a culture water body, can gradually release nutrients, and improves the effective utilization rate of the fertilizer.
Description
Technical Field
The invention belongs to the technical field of microbial application and fertilizer fermentation, and particularly relates to a Bacillus subtilis XDK06 with an anaerobic fermentation property, and application of the Bacillus subtilis in organic fertilizer fermentation.
Background
The bacillus subtilis is widely applied to the fermentation of organic fertilizers, but the oxygen consumption property of the existing bacillus subtilis determines that an oxygen consumption fermentation method is required to be adopted in the fermentation process of the organic fertilizers, so that an aeration facility is required in the fermentation process to meet the requirement of aeration, in addition, the fermentation process also needs to be frequently turned, otherwise, the effect of material decomposition cannot be achieved, and the labor intensity is high. Meanwhile, for the purpose of oxygen-consuming fermentation, the requirements on fermentation conditions such as initial water content, pH value, ambient temperature and the like are strict, materials also need to have certain permeability, and the whole fermentation process also needs to be kept at a high temperature of about 65 ℃ or even higher. In addition, the fertilizers formed by the fermentation method are all sinking, and when the fertilizers are applied to aquaculture water, the fertilizers can sink to the bottom of the pond immediately, so that the fertilizer efficiency is greatly reduced, and even a pollution source at the bottom of the pond can be formed, thereby limiting the application of the organic fertilizers in aquaculture. The fermentation process of the fermentation method can also convert a large amount of carbon and nitrogen nutrients in the organic fertilizer into ammonia gas and carbon dioxide, so that the ammonia gas and the carbon dioxide are volatilized into air, the production environment is severe due to odorous weather, equipment is easy to corrode and damage, the nutrient loss of the fertilizer can be caused, and energy is wasted.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a Bacillus subtilis XDK06 with anaerobic fermentation property, which is used for organic fertilizer fermentation, can obtain a floating fertilizer, greatly reduce labor intensity, reduce the nutrient loss of the fertilizer, improve the fermentation production environment and simplify the fermentation process of the fertilizer.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a Bacillus subtilis XDK06 is preserved in Guangdong province microorganism culture collection center in 10-30 months in 2020, with the preservation number of GDMCC No: 61260, the preservation address is No. 59 building of No. 100 Dazhong Jie-Lu, Guangzhou city.
As another aspect of the present invention, the present invention also provides a method for culturing the Bacillus subtilis XDK06, comprising the steps of: inoculating Bacillus subtilis XDK06 in a culture medium with pH value of 5.0-9.0, and controlling air volume at 30-50 deg.C and 30-200L/h-1And the stirring rate is 30 to 200r/min-1Culturing for 5-70 h under the condition of (1).
As a further optimization of the scheme, the inoculation amount of the bacillus subtilis XDK06 is 1-10%.
As a further optimization of the scheme, the culture medium comprises the following components in parts by weight: 10-30 parts of glucose, 10-40 parts of peptone, 3-10 parts of sodium chloride, 0.2-1 part of beef extract and 10-30 parts of agar.
As a further aspect of the invention, the invention also provides the use of said Bacillus subtilis XDK06 in the fermentation of organic fertilizers. The bacillus subtilis XDK06 can ferment decomposed organic matters under anaerobic conditions, the fermented fertilizer has low loss rate of nutrient components and buoyancy in water, the fermentation temperature can be continuously kept constant at 30-50 ℃ in the whole fermentation process, aeration is not needed, frequent pile turning is not needed, and pile turning is only needed once every 10-30 days.
As a further aspect of the invention, the invention also provides a fermentation method of the organic fertilizer, which takes the bacillus subtilis XDK06 as zymophyte.
As a further optimization of the scheme, the fermentation method is to perform solid state fermentation under anaerobic conditions.
As a further aspect of the present invention, the present invention also provides an organic nutrient which is an anaerobic fermentation product of Bacillus subtilis XDK06 mixed with an organic material.
As further optimization of the scheme, the organic material comprises the following components in parts by weight: 30-70 parts of animal waste, 0-30 parts of mushroom residues and 0-30 parts of rice bran. Wherein, the animal manure comprises but is not limited to chicken manure, duck manure, goose manure, cow manure, sheep manure, pig manure and the like.
As a further optimization of the above scheme, the preparation method of the organic nutrient comprises the following steps: uniformly mixing an organic material and bacillus subtilis XDK06 to obtain an anaerobic fermentation system, and carrying out anaerobic fermentation on the anaerobic fermentation system at the temperature of 30-50 ℃ for 20-90 days to obtain the organic nutrient, wherein the initial water content of the organic material is 50-60%, and the pH value of the anaerobic fermentation system is 4.5-7.5.
As a further optimization of the scheme, the addition amount of the bacillus subtilis XDK06 is 0.3-5% of the weight of the organic materials.
Through a plurality of tests, the invention discovers that the fermentation temperature of the organic materials in the whole fermentation process can be maintained at 30-50 ℃ by fermenting under the conditions, and the fluctuation of the temperature from the 5 th day becomes small and tends to be stable. After 20-90 days of the constant medium temperature solid fermentation, the organic materials can be fully decomposed, the particles of the organic materials are refined, the organic materials have suspension property in water, and the loss rate of nutrient components in the organic materials is low. After the organic nutrient is applied to a water body, the organic nutrient does not sink to the water bottom immediately, but is suspended in the water and gradually releases nutrients, so that the growth of beneficial algae in the water body is promoted, and the organic nutrient can also be used as an attachment of beneficial bacteria in the water body, and is beneficial to the implantation growth of the beneficial bacteria in the water body.
In some embodiments, the organic material is mixed with bacillus subtilis XDK06 uniformly and piled, the piled material is controlled within 100 tons/pile, and when the room temperature is lower than 20 ℃, the piled material is covered by a film. Turning the pile once every 10-30 days in the fermentation process, and turning the pile 1-2 times in the whole fermentation process.
As a further aspect of the invention, the invention also provides the application of the organic nutrient in a fertilizer for aquaculture or a fertilizer for planting.
As a further aspect of the invention, the invention also provides a fertilizer for aquaculture, which comprises the organic nutrient.
As a further aspect of the invention, the invention also provides a fertilizer for the crop industry comprising the organic nutrient.
Compared with the prior art, the invention has the beneficial effects that: the bacillus subtilis XDK06 provided by the invention has anaerobic fermentation property, and can ferment and decompose organic materials under anaerobic conditions. The fermentation method provided by the invention does not need aeration, the fermentation temperature can be kept at the medium temperature of 30-50 ℃, the fermentation process is environment-friendly, safe and energy-saving, the environmental pollution is small, the whole fermentation process only needs to be turned and piled for 1-2 times, the labor intensity is greatly reduced, organic materials can be thoroughly decomposed, the nutrient components of the organic materials are retained to a greater extent, the produced fertilizer has buoyancy, the fertilizer cannot be immediately submerged into the water bottom after being applied to a culture water body, the nutrients can be gradually released, the effective utilization rate of the fertilizer is improved, and the organic fertilizer is suitable for aquaculture.
Drawings
FIG. 1 is a gene sequence of Bacillus subtilis XDK 06;
fig. 2 is a continuation of fig. 1.
Detailed Description
One of the purposes of the invention is to provide a Bacillus subtilis XDK06 with anaerobic fermentation property, which can ferment and decompose organic materials under anaerobic conditions, thereby obtaining a floating organic fertilizer, simplifying the fermentation process of the organic fertilizer, reducing the fermentation cost and improving the application effect of the organic fertilizer in aquaculture water. The invention also provides a culture method of the bacillus subtilis. The invention also provides a method for preparing an organic fertilizer by applying the bacillus subtilis. By adopting the technical scheme, the limitation of the existing organic fertilizer fermentation is fundamentally solved, the nutrient content of the organic fertilizer is improved, and the organic fertilizer has buoyancy in water, so that the organic fertilizer is more suitable for being used as a fertilizer for aquaculture.
For the purpose of promoting an understanding of the invention, reference will now be made to the following examples which are intended to be further illustrative.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, the percentages are by weight unless otherwise specified.
Example 1 Strain acclimatization and screening
(1) Preparing acclimatization culture medium
Firstly, the chicken manure, the mushroom residues and the rice bran are subjected to pretreatment such as air drying, impurity removal, crushing and the like, and then the chicken manure, the mushroom residues and the rice bran are uniformly mixed according to the weight ratio of 30-50: 20-30 for later use;
(2) preparing the strain to be domesticated
Selecting 12 strains of yeast, 16 strains of bacillus subtilis and 8 strains of lactobacillus acidophilus as strains to be domesticated;
(3) acclimatized environmental conditions
An anaerobic fermentation environment with a water content of 50-60%, a pH value of 4.5-7.5 and a temperature of 30-50 ℃;
(4) acclimatization process
And (2) filling 1 kg of the domestication culture medium into a 2L triangular flask, adjusting the water content and the pH value according to the conditions, then respectively inoculating 1-5% of the strains to be domesticated, covering a rubber plug, putting the strains into an incubator in the temperature range, culturing for 3-10 days, taking out, and detecting two indexes of the weight of the water-insoluble substances and the suspension of the materials. Taking the first five groups with the least water insoluble substances and the best material suspensibility as target strains for continuous passage, taking 0.8 kg of domestication culture medium and 0.2 kg of target strain fermentation materials for each generation, carrying out repeated domestication passage for 20 generations, finally domesticating a strain which is most suitable for anaerobic fermentation and has the best material suspensibility, namely a strain which is most suitable for anaerobic fermentation organic fertilizer, naming the strain as XDK06, sending the strain to molecular genetic laboratory of southern China agricultural university for DNA sequencing, and finally determining that the strain is a Bacillus subtilis strain which is preserved in Guangdong province microorganism strain preservation center in 10 months and 30 days 2020 by the inventor, naming the strain as Bacillus subtilis XDK06, wherein the preservation number is MCC No: 61260.
a group of data with the best algebraic expression corresponding to the acclimatization process and the acclimatization effect in this embodiment is shown in the following table:
example 2
The preparation method of the organic nutrient is carried out indoors at the room temperature of 20-25 ℃, and comprises the following preparation steps: uniformly mixing chicken manure, mushroom residues and rice bran in a weight ratio of 50:25:25, then piling, controlling the weight of each pile to be within 100 tons, then uniformly adding bacillus subtilis XDK06 to obtain an anaerobic fermentation system, wherein the adding amount of the bacillus subtilis XDK06 is 2 percent of the weight of an organic material, carrying out anaerobic fermentation on the anaerobic fermentation system at the temperature of 30-50 ℃ for 60 days, controlling the initial water content of the material to be 50-60 percent, controlling the pH value of the anaerobic fermentation system to be 4.5-7.5, turning the piles once every 20 days in the fermentation process, and turning the piles for 2 times in the whole fermentation process. In the fermentation process, as the adopted fermentation raw materials (namely the chicken manure, the mushroom residue and the rice bran) are subjected to pretreatment such as air drying, impurity removal, crushing and the like, the material particles are very small, and therefore, oxygen in the air cannot enter the stockpile after the stockpile, so that the method of the embodiment piles the materials, and the anaerobic fermentation condition of the bacillus subtilis XDK06 can be met; other embodiments are the same.
Example 3
A method for preparing organic nutrient is carried out indoors, the room temperature is higher than 25 ℃, and the preparation steps are as follows: uniformly mixing chicken manure and mushroom residues according to a weight ratio of 70:30, then piling, controlling the weight of each pile within 100 tons, then uniformly adding bacillus subtilis XDK06 to obtain an anaerobic fermentation system, controlling the adding amount of the bacillus subtilis XDK06 to be 5% of the weight of an organic material, carrying out anaerobic fermentation on the anaerobic fermentation system at 30-50 ℃ for 20 days, controlling the initial water content of the material to be 50-60%, controlling the pH value of the anaerobic fermentation system to be 4.5-7.5, and turning the piles once on the 10 th day of fermentation.
Example 4
A method for preparing organic nutrient is carried out indoors at room temperature lower than 20 ℃, and comprises the following steps: uniformly mixing chicken manure, mushroom residues and rice bran in a weight ratio of 60:25:15, then piling, controlling the weight of each pile to be within 100 tons, then uniformly adding bacillus subtilis XDK06 to obtain an anaerobic fermentation system, controlling the adding amount of bacillus subtilis XDK06 to be 0.3 percent of the weight of organic materials, carrying out anaerobic fermentation on the anaerobic fermentation system at the temperature of 30-50 ℃ for 90 days, controlling the initial water content of the materials to be 50-60 percent, controlling the pH value of the anaerobic fermentation system to be 4.5-7.5, covering the piles with films, turning the piles once every 30 days in the fermentation process, and turning the piles for 2 times in the whole fermentation process.
Examples of effects
Experimental group 1: the chlorella is cultured in a water body of 3 cubic meters, clean Xijiang water is adopted, the environment temperature is 26-32 ℃ during the test period, and the water temperature is about 30 ℃. Adding 25 g of the organic nutrient of example 2 and 2 g of urea into a water body, observing the culture process through microscope examination, and multiplying the culture process by the microscope: eyepiece: 10 ×, objective: 40 ×, repeat three times, results are shown in the following table:
control group: the test method is the same as that of experimental group 1 except that 25 g of the organic nutrient of example 2 of experimental group 1 was changed to 25 g of chicken manure, and the other conditions were the same. The culture process is observed by microscope examination, and the culture process is microscopically multiplied: eyepiece: 10 ×, objective: 40 ×, repeat three times, results are shown in the following table:
from the above observation results, it can be seen that the organic nutrient of example 2 of the present invention more promoted the growth of chlorella than the control group. The inventors also performed the same tests on the organic nutrients of example 3 and example 4, and the test results were close to those of example 2, and are not described herein again.
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.
SEQUENCE LISTING
<110> New bioproduct Co Ltd of He shan City
<120> bacillus subtilis XDK06 and application thereof
<130> 20201118
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1145
<212> DNA
<213> unknown
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gacccgggcg ggcgtgctat acatgcaagt cgagcggaca gatgggagct tgctccctga 60
tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc 120
cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaaa cataaaaggt 180
ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240
ctcaccaagg caacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300
acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360
tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420
gaagaacaag taccgttcga atagggcggt accttgacgg tacctaacca gaaagccacg 480
gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540
gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600
gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660
tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720
gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780
cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840
acgcattaag cactccgcct gggggagtac ggtcgcaaga ctgaactcaa aggaattgac 900
gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960
caggtcttga acttcctctg acaatcctag agataggacg tcccccttcg ggggcagagt 1020
gacaagggtg gtgcattgct tgtcgtccgc ctcgagtccg tgaagatgtt gggctagtcc 1080
cccgcaccca gcgcaaccct tgaatcttag tttgccagct atttcagttg gcactcctca 1140
Claims (10)
1. A bacillus subtilis XDK06 which is preserved in Guangdong province microorganism culture collection center in 10 and 30 months in 2020 and has the preservation number of GDMCC No: 61260, the preservation address is No. 59 building of No. 100 Dazhong Jie-Lu, Guangzhou city.
2. The method for culturing Bacillus subtilis XDK06 according to claim 1, wherein the Bacillus subtilis XDK06 is cultured in the presence of a culture mediumThe method comprises the following steps: inoculating Bacillus subtilis XDK06 in a culture medium with pH value of 5.0-9.0, and controlling air volume at 30-50 deg.C and 30-200L/h-1And the stirring rate is 30 to 200r/min-1Culturing for 5-70 h under the condition of (1); preferably, the inoculation amount of the bacillus subtilis XDK06 is 1-10%; preferably, the culture medium comprises the following components in parts by weight: 10-30 parts of glucose, 10-40 parts of peptone, 3-10 parts of sodium chloride, 0.2-1 part of beef extract and 10-30 parts of agar.
3. Use of bacillus subtilis XDK06 as claimed in claim 1 in the fermentation of organic fertilizers.
4. A method for fermenting an organic fertilizer, which comprises fermenting Bacillus subtilis XDK06 according to claim 1.
5. The fermentation process of claim 4, wherein the fermentation process is a solid state fermentation under anaerobic conditions.
6. An organic nutrient which is an anaerobic fermentation product of bacillus subtilis XDK06 as defined in claim 1 mixed with an organic material; preferably, the organic material comprises the following components in parts by weight: 30-70 parts of animal waste, 0-30 parts of mushroom residues and 0-30 parts of rice bran.
7. The method for preparing the organic nutrient according to claim 6, comprising the steps of: uniformly mixing an organic material and bacillus subtilis XDK06 to obtain an anaerobic fermentation system, and carrying out anaerobic fermentation on the anaerobic fermentation system at the temperature of 30-50 ℃ for 20-90 days to obtain the organic nutrient, wherein the initial water content of the organic material is 50-60%, and the pH value of the anaerobic fermentation system is 4.5-7.5.
8. Use of the organic nutrient of claim 6 in a fertilizer for aquaculture or a fertilizer for farming.
9. An aquaculture fertilizer comprising the organic nutrient of claim 6.
10. A fertilizer for use in crop cultivation, comprising the organic nutrient of claim 6.
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