CN110305819B - Efficient feather degradation strain and application thereof - Google Patents

Efficient feather degradation strain and application thereof Download PDF

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CN110305819B
CN110305819B CN201910757813.8A CN201910757813A CN110305819B CN 110305819 B CN110305819 B CN 110305819B CN 201910757813 A CN201910757813 A CN 201910757813A CN 110305819 B CN110305819 B CN 110305819B
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feather
culture medium
proline
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culturing
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CN110305819A (en
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谢婧婧
宋天顺
李洋
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Nanjing Tech University
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    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F1/00Fertilisers made from animal corpses, or parts thereof
    • C05F1/005Fertilisers made from animal corpses, or parts thereof from meat-wastes or from other wastes of animal origin, e.g. skins, hair, hoofs, feathers, blood
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00Other organic fertilisers
    • C05F11/08Organic fertilisers containing added bacterial cultures, mycelia or the like
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00Preparation of nitrogen-containing organic compounds
    • C12P13/04Alpha- or beta- amino acids
    • C12P13/24Proline; Hydroxyproline; Histidine
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/20Flavobacterium
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/20Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00Technologies for solid waste management
    • Y02W30/40Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention discloses a feather high-efficiency degradation strain, which is classified and named as Flavobacterium tirrenicum (Flavobacterium tirrenicum), has a strain number of X-Y4, is preserved in China center for type culture Collection, and has a preservation number of CCTCC NO: m2019286, preservation date of 2019, 4 months and 23 days. The invention also discloses application of the Chryseobacterium in preparation of proline by degrading feathers. The strain bred by the invention can effectively solve the problem of recycling of waste feathers, can be used as a liquid fertilizer for farmland irrigation, realizes the comprehensive utilization of biomass resources and simultaneously reduces the pollution of poultry cultivation to the environment. The bacterium has strong degradation capability and short fermentation period, and the proline content in the fermentation liquor is more than 92%. The proline-rich fermentation liquor can be prepared into liquid fertilizer, has the effects of promoting plant growth, relieving salt stress and acid stress and the like, and enhances the nutritive value and the utilization rate of the fertilizer.

Description

Efficient feather degradation strain and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a feather degrading bacterium and application thereof.
Background
The large consumption of animals generates a lot of feather wastes, and the traditional treatment method mainly comprises landfill, incineration and gas generation, thus causing great harm to the environment. Earlier researches on the preparation of animal feed by using poultry feathers have achieved application and development on a certain scale, but 90% of keratin in the feathers is not efficiently utilized, so that waste of available resources is caused. Keratin has been used in the cosmetics industry as a major component of feathers, or is utilized as a component of composite materials, fibrous products. The keratin resources in China are extremely rich, and especially in modern agriculture, large-scale poultry cultivation generates a large amount of keratin waste, wherein the feather waste yield is the largest, and the annual yield reaches more than 70 ten thousand tons. According to the measurement, the crude protein content of the feather keratin is about more than 80 percent, the amino acid content is more than 70 percent, and the feather keratin also contains macroelements, microelements, vitamins and some unknown growth factors. Therefore, the feather keratin waste has wide application after proper treatment.
Keratin is a complex three-dimensional network structure due to the cross-linking of a large number of disulfide bonds, has good stability and insolubility, and is difficult to hydrolyze by a common method, so that the research hot tide of a feather degradation method is initiated. The common approaches at present are physical degradation, chemical degradation and biotechnology. Although physical and chemical methods are studied earlier, the method has the defects of destroying the amino acid structure, single product, poor product stability and the like. Compared with the traditional physical and chemical methods, the method for treating the waste feather keratin by the biotechnology method has the advantages of mild reaction conditions, high product recovery rate, environmental friendliness, energy conservation and the like. The biotechnology method mainly comprises an enzymatic hydrolysis method and a microbial degradation method. The microbial degradation method is characterized in that the keratin is degraded into polypeptide and amino acid by using the characteristic that the keratinase generated by microorganisms is used, so that the possibility is provided for deep utilization of feathers. Proline is a soluble organic substance, can regulate the osmotic potential in cytoplasm, and plays an important role in the defense mechanism of stressed cells. Proline is used to protect cells against the adverse effects of salt stress, mainly by maintaining osmotic balance, stabilizing subcellular structures such as membranes and proteins, and scavenging reactive oxygen species. Proline degradation can provide a carbon source, a nitrogen source, and energy. Under environmental stress, the stress resistance of plants can be improved by externally applying proline.
No report of bacterial strains capable of efficiently degrading feathers into high-purity proline is found in the prior art.
Disclosure of Invention
The invention provides a bacterial strain capable of efficiently degrading waste feathers, which can degrade feathers into high-purity proline.
The invention also aims to solve the technical problem of providing the application of the feathers in preparing proline by fermentation.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a feather high-efficiency degradation strain is classified and named as Flavobacterium tirrenicum with a strain number of X-Y4, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019286, the preservation date is 2019, 4 and 23, and the preservation address is Wuhan university in Wuhan, China.
The Flavobacterium tirrenicum X-Y4 capable of efficiently degrading the feathers has the following biological characteristics of bacilliform thallus, no spore production, no motility, negative gram staining and yellow colony formation on a nutrient agar culture medium (shown in figure 1).
The screening process comprises the following steps:
a. preparation of selection Medium
b. Preliminary screening of feather degradation fungus
c. Rescreening of feather degradation bacteria
The step a may further include: preparing a feather solid culture medium and preparing a feather liquid culture medium.
The step b may further include: weighing 5g of soil from the air-dried soil sample of the pigeon factory, adding the soil into 50ml of sterile water for enrichment culture, and culturing for 12h under the culture condition of 30 ℃ and 160 r/min; collecting the enrichment culture solution according to the formula 10-1、10-2、 10-3、10-4、10-5、10-6、10-7、10-8And (3) performing gradient dilution on the feather solid culture medium, coating the feather solid culture medium, performing purification and streak culture for 5 times when a single colony grows out, and re-screening the feather liquid culture medium to obtain the efficient feather degrading bacteria.
The step c may further include: inoculating the screened strain into 100ml of feather liquid culture medium, respectively culturing at 30 ℃ and 160r/min for 3d, and determining the proline production capacity of the strain.
The strain identification method comprises the following steps:
16sRNA sequence analysis: the PCR amplification adopts a bacterial 16sRNA universal primer:
27F5’-AGAGTTTGATCMTGGCTCAG-3’
1492R5’-GGTTACCTTGTTACGACTT-3’
PCR reaction (50. mu.L): 10 Xamplification buffer (5. mu.L), genomic DNA (1. mu.L), dNTP (2. mu.L), primer F (2. mu.L), primer R (2. mu.L), Taq DNA polymerase (0.25. mu.L), and deionized water (37.75. mu.L). Reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min, deformation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 25 cycles, and full extension at 72 ℃ for 10 min.
Most of the 16S rRNA sequence was determined as shown in SEQ ID No: 1 is shown.
The strain X-Y4 has the following properties:
1. morphological characteristics of colonies:
after culturing for 1-2 days at 30 ℃ on an LB solid culture medium, the vegetative cells are observed to be single cells and rod-shaped by a microscope. The cells were cultured in the above medium at 30 ℃ for 12 hours to allow mass growth. The bacterial colony is golden yellow, round, smooth in surface, neat in edge, viscous and convex in the middle.
2. Physiological and biochemical characteristics:
TABLE 1 physiological and biochemical characteristics of the strains
Figure BDA0002169302720000031
3. 16S rDNA sequence analysis
27F 5’-AGAGTTTGATCMTGGCTCAG-3’
1492R 5’-GGTTACCTTGTTACGACTT-3’
PCR reaction (50. mu.L): 10 Xamplification buffer (5. mu.L), genomic DNA (1. mu.L), dNTP (2. mu.L), primer F (2. mu.L), primer R (2. mu.L), Taq DNA polymerase (0.25. mu.L), and deionized water (37.75. mu.L). Reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min, deformation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 25 cycles, and full extension at 72 ℃ for 10 min.
The 16S rDNA major sequence is determined as shown in SEQ ID No: 1 is shown. The sequence was aligned with the base sequence using BLAST from NCBI website to construct a phylogenetic tree based on the 16S rRNA full sequence. The results show that: the strain and the golden yellow bacillus reach 98 percent of homology. Therefore, Flavobacterium tirnenicum (Flavobacterium tirnenicum) is considered to be used in the invention, and the site is Flavobacterium tirnenicum X-Y4.
The application of the strain in preparing proline by degrading feathers is also within the protection scope of the invention.
The method for preparing the fertilizer by degrading the feathers by the bacterial strain comprises the steps of activating the bacterial strain, coating the bacterial strain on a feather solid culture medium, and culturing for 16-24 hours (preferably 18 hours) at the temperature of 30-40 ℃; selecting strains on the feather solid culture medium, purifying and streaking the strains on a fresh feather solid culture medium, and culturing the strains for 16-24 hours (preferably 18 hours) at the temperature of 30-40 ℃ to form single colonies; selecting a single colony on a feather solid culture medium, inoculating the single colony in an LB liquid culture medium, and culturing at 25-40 ℃ for 16-24 h (preferably to OD)6000.6), inoculating into a feather liquid culture medium, inoculating with 2% v/v, and culturing at 25-40 deg.C (preferably 35 deg.C) for 60-80 hr (preferably 72 hr) to obtain proline-containing fermentation broth.
Wherein, the formula of the feather solid medium is preferably as follows: 10g of feather, 1g of sodium chloride, 1g of dipotassium hydrogen phosphate, 0.4g of monopotassium phosphate, 20g of agar powder and 1000ml of water.
Wherein, the formula of the LB liquid culture medium is preferably as follows: 10.0g/L of peptone, 5.0g/L of yeast powder, 10.0g/L of sodium chloride and 1000ml of water.
Wherein, the formula of the feather liquid culture medium is preferably as follows: 10g of feather, 1g of sodium chloride, 1g of dipotassium phosphate, 0.4g of monopotassium phosphate and 1000ml of water.
Wherein, in the final fermentation liquor, the concentration of proline is more than 1.0g/L, and the mass percentage of proline in the degraded amino acid product is more than 92%.
The fermentation product of the strain contains rich available amino acids, the degradation efficiency is high, the fermentation period is short, the loss of most amino acids is avoided, the utilization rate of feather degradation products is improved, and the proline content in the fermentation liquor is more than 92%. Proline is a main osmoregulation substance accumulated in plants under adverse stresses such as drought, high temperature, high salinity, freezing, ultraviolet irradiation, heavy metals and the like. The fermentation liquor rich in proline can be prepared into liquid fertilizer, and has the effects of promoting plant growth, relieving salt stress, relieving acid stress and the like.
The application of the strain in preparing fertilizer by degrading feathers is also within the protection scope of the invention.
The method for preparing the fertilizer by degrading the feathers by the bacterial strain comprises the steps of activating the bacterial strain, coating the bacterial strain on a feather solid culture medium, and culturing for 16-24 hours (preferably 18 hours) at the temperature of 30-40 ℃; selecting strains on the feather solid culture medium, purifying and streaking the strains on a fresh feather solid culture medium, and culturing the strains for 16-24 hours (preferably 18 hours) at the temperature of 30-40 ℃ to form single colonies; selecting a single colony on a feather solid culture medium, inoculating the single colony in an LB liquid culture medium, and culturing at 25-40 ℃ for 16-24 h (preferably to OD)6000.6), inoculating into a feather liquid culture medium, culturing for 60-80 hours (preferably 72 hours) at 25-40 ℃ (preferably 35 ℃) by an inoculation amount of 2% v/v to obtain fermentation liquor containing proline, and centrifuging the fermentation liquor to obtain supernatant serving as a liquid fertilizer.
The obtained fermentation liquor is preferably subjected to solid-liquid separation to remove thalli, and a clear solution is reserved as a liquid fertilizer.
Wherein, the formula of the feather solid medium is preferably as follows: 10g of feather, 1g of sodium chloride, 1g of dipotassium hydrogen phosphate, 0.4g of monopotassium phosphate, 20g of agar powder and 1000ml of water.
Wherein, the preferable formula of the LB liquid culture medium is as follows: 10.0g/L of peptone, 5.0g/L of yeast powder, 10.0g/L of sodium chloride and 1000ml of water.
Wherein, the formula of the feather liquid culture medium is preferably as follows: 10g of feather, 1g of sodium chloride, 1g of dipotassium phosphate, 0.4g of monopotassium phosphate and 1000ml of water.
The concrete application method of the prepared fertilizer comprises the following steps: root irrigation is carried out for 1 time every 6-8 days, and the application amount is 0.2-0.6L/m2And 3-5 times, and the application concentration is 5-15 mmol/L calculated by the concentration of proline.
The fertilizer can promote plant growth and relieve the stress of salt and acid in soil.
Has the advantages that: the efficient feather degradation and the preparation of the feather degradation product have the following advantages:
the method selects the bacteria Flavobacterium tirrenicum X-Y4 with the capability of degrading the feather from the soil, and takes the feather degrading bacteria Flavobacterium tirrenicum X-Y4 as strains to ferment to obtain the feather degradation product, wherein the feather degradation product contains high-purity proline, can be used as an amino acid liquid fertilizer, relieves the salt stress action and relieves the stress action of acid soil on plants, and can effectively improve the yield of the pakchoi.
Drawings
FIG. 1 shows the colony morphology on LB plates of X-Y4.
FIG. 2 is a gram stain of X-Y4.
FIG. 3 is a schematic diagram of an example of phylogenetic analysis of the 16S rRNA sequence of X-Y4.
FIG. 4 is a bar graph of feather addition versus soluble protein concentration.
FIG. 5 is a bar graph of shaker rotation speed versus soluble protein concentration.
Figure 6 is a bar graph of initial pH versus soluble protein concentration.
FIG. 7 is a bar graph of inoculum size versus soluble protein concentration.
Figure 8 is a bar graph of temperature versus soluble protein concentration.
FIG. 9 is a graph showing the effect of XY-4 fermentation for 72h on feather degradation.
FIG. 10 is a line graph of feather degradation over 72 hours.
Detailed Description
The foregoing aspects of the present invention are described in further detail below by way of examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above aspects of the present invention are within the scope of the present invention.
Example 1 screening of Chryseobacterium X-Y4
(1) Sample source: and (5) piling up the rotten feather field soil sample in the pigeon raising field for a long time.
(2) Strain screening:
the screening process comprises the following steps:
a. preparation of selection Medium
b. Preliminary screening of feather degradation fungus
c. Rescreening of feather degradation bacteria
The step a may further include: preparing a feather solid culture medium and preparing a feather liquid culture medium.
The step b may further include: weighing 5g of soil from the air-dried soil sample of the pigeon factory, adding the soil into 50ml of sterile water for enrichment culture, and culturing for 12h under the culture condition of 30 ℃ and 160 r/min; collecting the enrichment culture solution according to the formula 10-1、10-2、 10-3、10-4、10-5、10-6、10-7、10-8And (3) performing gradient dilution on the feather solid culture medium, coating the feather solid culture medium, performing purification and streak culture for 5 times when a single colony grows out, and performing primary screening on a feather liquid culture medium to obtain high-efficiency feather degrading bacteria.
The step c may further include: inoculating the screened strains into 100ml of feather liquid culture medium, respectively culturing at 30 ℃ and 160r/min for 3d, measuring the proline content in the fermentation liquor (see table 2), and screening out the strain X-Y4 with the highest proline yield.
TABLE 2 proline content in fermentation broths of different strains
Figure BDA0002169302720000061
Figure BDA0002169302720000071
(3) Colony morphology observation of X-Y4 strain
The screened X-Y4 strain was cultured on a feather solid medium at 30 ℃ for 1 day and then gram-stained, and the vegetative cells were observed as single cells, rods, by a microscope, as shown in FIG. 2. The cells were cultured in the above medium at 30 ℃ for 12 hours to allow mass growth. The colony is golden yellow, round, smooth in surface, neat in edge, viscous, and convex in the middle, as shown in fig. 2.
Example 2: and (5) identifying strains.
16sRNA sequence analysis: the PCR amplification adopts a bacterial 16sRNA universal primer:
27F5’-AGAGTTTGATCMTGGCTCAG-3’
1492R5’-GGTTACCTTGTTACGACTT-3’
dNTP (2. mu.L), primer F (2. mu.L), primer R (2. mu.L), Taq DNA polymerase (0.25. mu.L), and deionized water (37.75. mu.L). Reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min, deformation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 25 cycles, and full extension at 72 ℃ for 10 min.
The 16S rDNA major sequence is determined as shown in SEQ ID No: 1 is shown. The sequence was aligned with the base sequence using BLAST from NCBI website to construct a phylogenetic tree based on the 16S rRNA full sequence. The results show that: the strain and the golden yellow bacillus reach 98 percent of homology. Therefore, Flavobacterium tirnenicum (Flavobacterium tirnenicum) is considered to be used in the invention, and the site is Flavobacterium tirnenicum X-Y4.
Example 3: optimization of culture conditions of feather degradation strain
(1) The strain is as follows: example 1 selected Strain Flavobacterium tirrenicum X-Y4
(2) The method comprises the following steps:
and (3) adopting a single-factor test to investigate the influence of the feather addition amount, the table rotating speed, the initial pH, the inoculation amount and the fermentation temperature of the strain on the yield of the soluble protein.
a. Preferred test for feather addition
The fermentation conditions comprise that the feather liquid culture medium is filled with 100mL/250mL of liquid, the inoculum size is 2% (v/v), the temperature is 30 ℃, the speed is 160r/min, the pH value is 7.0, and the feather is fermented in a shake flask for 72 hours, and the feather filling amount is 0.5, 1.0g, 1.5g, 2.0g, 2.5g and 3.0g respectively.
b. Preferred experiment of the rotational speed of the rocking platforms
The feather adding amount is measured to obtain the optimal result determined by the test, other fermentation conditions are that the feather liquid culture medium is filled with 100mL/250mL of liquid, the inoculation amount is 2% (v/v), the temperature is 30 ℃, the pH value is 7.0, the fermentation is carried out for 72 hours in a shake flask, the rotating speeds of a shaking table are respectively 140r/min, 160r/min, 180r/min, 200r/min, 220r/min and 240r/min, and the influence of the rotating speeds of the shaking table on the production of soluble protein by the strain is inspected.
c. Preferred test of initial pH
And (3) determining the optimal result of the feather addition amount and the rotating speed of a shaking table, wherein other fermentation conditions comprise 100mL/250mL of feather liquid culture medium liquid loading, 2% (v/v) of inoculum size, 30 ℃ and 72h of shaking flask fermentation, and through early-stage tests, primarily adjusting the initial pH to be 8.0, 8.5, 9.0, 9.5 and 10.0 respectively to investigate the influence of the pH on soluble protein of the strain.
d. Preferred test of the amount of inoculation
The optimal results determined by the tests are taken for the feather addition amount, the shaking table rotating speed and the initial pH, other fermentation conditions are that the feather liquid culture medium is filled with 100mL/250mL, the temperature is 30 ℃, shaking bottle fermentation is carried out for 72 hours, the inoculation amounts are 1%, 2%, 3%, 4% and 5%, respectively, and the influence of the shaking table rotating speed on the soluble protein production of the strain is inspected.
e. Preferred test of fermentation temperature
The optimal results determined by the tests are measured by the feather adding amount, the shaking table rotating speed, the initial pH and the inoculation amount, other fermentation conditions are that the feather liquid culture medium is filled with 100mL/250mL, the shaking table is used for fermentation for 72 hours, the soluble protein is measured after fermentation culture is respectively carried out in the shaking table at 25 ℃, 30 ℃, 35 ℃, 37 ℃ and 45 ℃, and the influence of the temperature on the soluble protein produced by the strain is inspected.
As can be seen from FIG. 4, the content of soluble protein is 122.35 + -3.53 μ g/ml at the maximum when the feather filling amount is 1 g; as can be seen from FIG. 5, the maximum soluble protein content was 121.32 + -20.76 μ g/ml at a rotation rate of 200 r/min; as can be seen from FIG. 6, the maximum soluble protein content was 162.71. + -. 35.51. mu.g/ml at an initial pH of 9.0; as can be seen from FIG. 7, the maximum soluble protein content was 168.56. + -. 7.82. mu.g/ml when the inoculum size was 2%; as can be seen from FIG. 8, the maximum soluble protein content was 173.27. + -. 2.95. mu.g/ml at a temperature of 35 ℃. In conclusion, the optimal fermentation conditions are as follows: the feather filling amount is 1g, the rotating speed is 200r/min, the initial pH is 9.0, the inoculation amount is 2 percent, the temperature is 35 ℃, and the maximum soluble protein content is 173.27 +/-2.95 mu g/ml.
Example 4: feather degradation experiment
Inoculating X-Y4 into feather liquid culture medium with an inoculum size of 2% (v/v), shaking flask culture medium liquid loading capacity of 100mL/250mL, pH of 9.0, feather addition amount of 1g/L, inoculum size of 2%, shaking flask fermentation at 35 deg.C and 200r/min for 72 h. The feather degradation phenomenon is observed by fermentation, and the culture medium is transparent and clear because the culture medium has no carbon-nitrogen source except the feathers. When the fermentation is carried out for about 12 hours, fine hairs on the main stems of the feathers fall off, and the culture medium is gradually turbid; when fermentation is carried out for 48 hours, the culture medium is turbid and light-tight, most of the feathers are degraded, and only the rest main stems are left; after 72h, the feather stem was also degraded in the medium. As shown in FIG. 9, after 72h of degradation, compared with the undegraded solution, the solution becomes turbid and golden yellow, large-particle feathers are not basically existed in the solution, and the feathers are basically and completely degraded. As shown in FIG. 10, the degradation rate in 72 hours can reach 95.72%.
Example 5: products of X-Y4 degrading pigeon feather
Activating feather degrading bacteria stored at-20 ℃, coating the feather degrading bacteria on a feather solid culture medium, and culturing for 18h at 30 ℃; selecting strains on the feather solid culture medium, purifying and streaking the strains on a fresh feather solid culture medium, and culturing the strains for 18 hours at the temperature of 30 ℃; and (3) selecting a single colony on the feather solid culture medium, inoculating the single colony in an LB liquid culture medium, culturing for 18h at 35 ℃, and culturing for 72h in the feather liquid culture medium at 35 ℃ to obtain fermentation liquor. Centrifuging the fermentation broth at 4 deg.C and 6000r/min for 15 min, collecting supernatant, and storing the supernatant in refrigerator at 4 deg.C. Dove hairs were used as feathers in this experiment.
The supernatant is the feather degradation product and can be used as an amino acid liquid fertilizer, wherein the content of proline is up to 92.58%.
TABLE 3 content of amino acids in the products of Pigeon Hair degradation
Figure BDA0002169302720000091
Figure BDA0002169302720000101
Example 6: X-Y4 product for degrading chicken feather
Activating feather degrading bacteria stored at-20 ℃, coating the feather degrading bacteria on a feather solid culture medium, and culturing for 18h at 30 ℃; selecting strains on the feather solid culture medium, purifying and streaking the strains on a fresh feather solid culture medium, and culturing the strains for 18 hours at the temperature of 30 ℃; and (3) selecting a single colony on the feather solid culture medium, inoculating the single colony in an LB liquid culture medium, culturing for 18h at 35 ℃, and culturing for 72h in the feather liquid culture medium at 35 ℃ to obtain fermentation liquor. Centrifuging the fermentation broth at 4 deg.C and 6000r/min for 15 min, collecting supernatant, and storing the supernatant in refrigerator at 4 deg.C. In this experiment, chicken feathers were used.
TABLE 4 amino acid content of the feather degradation product
Figure BDA0002169302720000102
Figure BDA0002169302720000111
Example 7: effect of different concentrations of feather degradation products on crop yield
The caliber of the flowerpot adopted in the experiment is 140mm, the height is 115mm, 650g of soil is filled in each pot, and the application amount of the fertilizer is 0.571 g.kg- 1NH4NO3,0.439g·kg-1KH2PO4,0.141g·kg-1KCl, uniformly mixing the fertilizer and the soil, putting the mixture into a pot, and adding a proper amount of water to completely soak the soil. Soaking the seeds in pure water overnight (4 ℃), selecting the plump and consistent seeds in plastic pots, sowing about 5 seeds in each pot, dispersing uniformly, culturing in a greenhouse until the seeds germinate, thinning the seedlings to 2 seedlings after germination, performing root irrigation treatment by using feather degradation product solutions with different volumes, wherein the concentration of proline is 5mmol/L, the proline concentration is 1 time every 7 days, and the dosage is 0.2L/m each time2Proline concentrations were 10mmol/L and 5mmol/L, respectively, for a total of 3 times, during which time water was applied daily, and water was maintained at about 75% field capacity using weight difference subtraction, and co-cultured for 30 d. The black leaf sunflower fan Chinese cabbage is taken as an experimental object, wherein:
control group: common soil plus fertilizer
Experimental group 1: common soil + fertilizer +0.2L/m2Feather degradation product (10mmol/L proline)
Experimental group 2: common soil + fertilizer +0.2L/m2Feather degradation product (5mmol/L proline)
Each treatment group was harvested on day 30 of plant growth, plants were cut along the roots, washed with water, and biomass was measured.
TABLE 5 Effect of different concentrations of feather degradation products on the characteristics and yield of Brassica campestris
Figure BDA0002169302720000112
Figure BDA0002169302720000121
As can be seen from Table 5, the feather degradation products can improve the plant height and fresh weight of the pakchoi to different degrees, the yield of the feather degradation product (10mmol/L proline) is increased by 21.10% compared with the yield of the feather degradation product which is not added, and the yield of the feather degradation product (5mmol/L proline) can still be increased by 7.07%, which shows that the feather degradation product has a promoting effect on the growth of the pakchoi, and the effect is more obvious when the concentration is higher in a certain range.
Example 8: effect of feather degradation products on crop yield at different application times
The caliber of the flowerpot adopted in the experiment is 140mm, the height is 115mm, 650g of soil is filled in each pot, and the application amount of the fertilizer is 0.571 g.kg- 1NH4NO3,0.439g·kg-1KH2PO4,0.141g·kg-1KCl, uniformly mixing the fertilizer and the soil, putting the mixture into a pot, and adding a proper amount of water to completely soak the soil. Soaking the seeds in pure water overnight (4 ℃), selecting the plump and consistent seeds in plastic pots, sowing about 5 seeds in each pot, uniformly dispersing the seeds, culturing the seeds in a greenhouse until the seeds germinate, thinning the seedlings to 2 seedlings after germination, performing root irrigation treatment by using feather degradation product solutions with different concentrations for 1 time every 4 days, 5 days and 7 days, wherein the dosage of each time is 0.2L/m2The proline concentration at the time of administration was 5 mmol/L. The culture was performed for a total of 5, 4, and 3 times, during which time watering was daily, and water was maintained at about 75% field capacity by weight difference subtraction, and co-cultured for 30 d. The black leaf sunflower fan Chinese cabbage is taken as an experimental object, wherein:
control group: common soil plus fertilizer
Experimental group 1: common soil + fertilizer +0.2L/m2Feather degradation products (applied every 4 d)Fertilizing once, 5 times in total)
Experimental group 2: common soil + fertilizer +0.2L/m2Feather degradation product (fertilization once every 5d, 4 times in total)
Experimental group 3: common soil + fertilizer +0.2L/m2Feather degradation product (fertilization once every 7d, 3 times in total)
Each treatment group was harvested on day 30 of plant growth, plants were cut along the roots, washed with water, and biomass was measured.
TABLE 6 influence of different application times of feather degradation products on the properties and yield of pakchoi
Experimental group Chinese cabbage plant height (cm) Pakchoi fresh weight (g/basin) Increased yield compared with control group
Control group 17.63±0.24 24.88±0.32 -
Experimental group 1 20.14±0.33 30.94±0.26 24.36%
Experimental group 2 19.67±0.21 29.32±0.29 17.85%
Experimental group 3 19.33±0.14 28.89±0.46 16.12%
As can be seen from Table 6, the application of feather degradation products for different times can improve the plant height and fresh weight of the pakchoi to different extents, the yield can be increased by 24.36% when the fertilizer is applied every 4 days compared with the application of no feather degradation product, the yield can be increased by 17.85% when the fertilizer is applied every 5 days, and the fertilizer can be increased by 16.12% when the fertilizer is applied every 7 days, which indicates that the feather degradation products have a promoting effect on the growth of the pakchoi, and the more the application times are, the more the gain is obvious in a certain range.
Example 9: effect of different application amounts of feather degradation products on crop yield.
The caliber of the flowerpot adopted in the experiment is 140mm, the height is 115mm, 650g of soil is filled in each pot, and the application amount of the fertilizer is 0.571 g.kg- 1NH4NO3,0.439g·kg-1KH2PO4,0.141g·kg-1KCl, uniformly mixing the fertilizer and the soil, putting the mixture into a pot, and adding a proper amount of water to completely soak the soil. Soaking the seeds in pure water overnight (4 ℃), selecting the plump and consistent seeds in plastic pots, sowing about 5 seeds in each pot, uniformly dispersing the seeds, culturing the seeds in a greenhouse until the seeds germinate, thinning the seedlings to 2 seedlings after germination, performing root irrigation treatment by using feather degradation product solutions with different concentrations for 1 time every 7 days, wherein the dosage of each time is 0.2L/m2、 0.4L/m2And 0.6L/m2The proline concentration at the time of administration was 5 mmol/L. A total of 3 times, during which watering was daily, water was maintained at about 75% field capacity using weight difference subtraction, and co-cultured for 30 d. The black leaf sunflower fan Chinese cabbage is taken as an experimental object, wherein:
control group 1: common soil plus fertilizer
Experimental group 1: common soil + fertilizer +0.2L/m2Feather degradation products
Experimental group 2: common soil + fertilizer +0.4L/m2Feather degradation products
Experimental group 3: common soil + fertilizer +0.6L/m2Feather degradation products
Each treatment group was harvested on day 30 of plant growth, plants were cut along the roots, washed with water, and biomass was measured.
TABLE 7 Effect of different amounts of feather degradation products on the identity and yield of Brassica rapa
Experimental group Chinese cabbage plant height (cm) Pakchoi fresh weight (g/basin) Increased yield compared with control group
Control group 17.33±0.35 24.79±0.24 -
Experimental group 1 20.12±0.37 29.59±0.26 19.36%
Experimental group 2 20.98±0.34 30.75±0.13 24.04
Experiment ofGroup
3 21.23±0.19 31.11±0.28 25.49%
As can be seen from Table 7, the application of feather degradation products in different amounts can increase the height and fresh weight of the pakchoi in different degrees, applying 0.2L/m2The yield of the feather degradation product is increased by 19.36 percent compared with the feather degradation product without feather, and 0.4L/m is applied2The feather degradation product can increase the yield by 24.04 percent and apply 0.6L/m2The feather degradation product can be fertilized by 25.49 percent, which shows that the feather degradation product has promotion effect on the growth of the pakchoi, and the effect is more obvious when the application amount is more in a certain range.
Example 10: the feather degradation product can relieve the stress action of saline-alkali soil on the pakchoi.
The caliber of the flowerpot adopted in the experiment is 140mm, the height is 115mm, 650g of soil is filled in each pot, and the application amount of the fertilizer is 0.571 g.kg- 1NH4NO3,0.439g·kg-1KH2PO4,0.141g·kg-1KCl, uniformly mixing the fertilizer and the saline-alkali soil, putting the mixture into a pot, and putting a proper amount of water into the pot to completely soak the soil. Soaking the seeds in pure water overnight (4 ℃), selecting the plump and consistent seeds in plastic pots, sowing about 5 seeds in each pot, uniformly dispersing the seeds, culturing the seeds in a greenhouse until the seeds germinate, thinning the seedlings to 2 seedlings after germination, performing root irrigation treatment by using feather degradation product solutions with different volumes, and performing root irrigation treatment for 1 time every 7 days with the dosage of 0.2L/m each time2Or 0.4L/m2The proline concentration at the time of administration was 15 mmol/L. A total of 3 times, during which watering was daily, water was maintained at about 75% field capacity using weight difference subtraction, and co-cultured for 30 d. The black leaf sunflower fan Chinese cabbage is taken as an experimental object, wherein:
control group: saline-alkali soil and fertilizer
Experimental group 1: saline-alkali soil + fertilizer +0.2L/m2Feather degradation products
Experimental group 2: saline-alkali soil + fertilizer +0.4L/m2Feather degradation products
Each treatment group was harvested on day 30 of plant growth, plants were cut along the roots, washed with water, and biomass was measured.
TABLE 8 influence of feather degradation products on the characteristics and yield of pakchoi in saline-alkali soil
Experimental group Chinese cabbage plant height (cm) Pakchoi fresh weight (g/basin) Increased yield compared with control group
Control group 11.86±0.31 17.61±0.47 -
Experimental group 1 17.32±0.23 21.96±0.42 24.71%
Experimental group 2 17.86±0.25 24.45±0.64 38.84%
As can be seen from Table 8, the application of the feather degradation products in the saline-alkali soil significantly increases the height and fresh weight of the pakchoi, and the application of 0.2L/m2Degradation products of feather and degradation without featherThe product yield is increased by 24.71 percent and 0.4L/m2The yield of the feather degradation product can be increased by 38.84%, which shows that the feather degradation product can also play a role in promoting growth of the pakchoi in the saline-alkali soil.
Example 11: the feather degradation product can relieve the stress action of acid soil on the pakchoi.
The caliber of the flowerpot adopted in the experiment is 140mm, the height is 115mm, 650g of soil is filled in each pot, and the application amount of the fertilizer is 0.571 g.kg- 1NH4NO3,0.439g·kg-1KH2PO4,0.141g·kg-1KCl, uniformly mixing the fertilizer and the acid soil, putting the mixture into a pot, and adding a proper amount of water to completely soak the soil. Soaking the seeds in pure water overnight (4 ℃), selecting the plump and consistent seeds in plastic pots, sowing about 5 seeds in each pot, uniformly dispersing the seeds, culturing the seeds in a greenhouse until the seeds germinate, thinning the seedlings to 2 seedlings after germination, performing root irrigation treatment by using feather degradation product solutions with different concentrations for 1 time every 7 days, wherein the dosage of each time is 0.2L/m2、0.4L/m2The proline concentration at the time of administration was 5 mmol/L. A total of 3 times, during which watering was daily, water was maintained at about 75% field capacity using weight difference subtraction, and co-cultured for 30 d. The black leaf sunflower fan Chinese cabbage is taken as an experimental object, wherein:
control group: acid soil and fertilizer
Experimental group 1: acid soil + fertilizer +0.2L/m2Feather degradation products
Experimental group 2: acid soil + fertilizer +0.4L/m2Feather degradation products
Each treatment group was harvested on day 30 of plant growth, plants were cut along the roots, washed with water, and biomass was measured.
TABLE 9 Effect of feather degradation products on acid soil pakchoi Properties and yield
Experimental group Chinese cabbage plant height (cm) Pakchoi fresh weight (g/basin) Increased yield compared with control group
Control group 11.33±0.48 17.89±0.24 -
Experimental group 1 16.32±0.23 22.86±0.33 27.78%
Experimental group 2 17.24±0.34 23.65±0.43 32.20%
As can be seen from Table 9, the application of the feather degradation products in the acid soil significantly increased the height and fresh weight of the pakchoi, and the application of 0.2L/m2The feather degradation product and the degradation product without feather are increased by 24.71 percent in comparison, 0.4L/m is applied2The yield of the feather degradation product can be increased by 38.84%, which shows that the feather degradation product can also play a growth promoting role on the pakchoi in acid soil.
Sequence listing
<110> Nanjing university of industry
<120> feather high-efficiency degradation strain and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1009
<212>DNA
<213> Flavobacterium terrestrium (Flavobacterium terrenicum)
<400>1
ctgttacggt caccgacttc aggtacccca gacttccatg gcttgacggg cggtgtgtac 60
aaggcccggg aacgtattca ccgcgccatg gctgatgcgc gattactagc gattccagct 120
tcatagagtc gagttgcaga ctccaatccg aactgagacc ggctttcgag atttgcatca 180
catcgctgtg tagctgccct ctgtaccggc cattgtatta cgtgtgtggc ccaaggcgta 240
agggccgtga tgatttgacg tcatccccac cttcctctct acttgcgtag gcagtctcac 300
tagagtcccc aacttaatga tggcaactag tgacaggggt tgcgctcgtt gcaggactta360
acctaacacc tcacggcacg agctgacgac aaccatgcag caccttgaaa attgcccgaa 420
ggaaggtcta tttctaaacc gatcaattcc catttaagcc ttggtaaggt tcctcgcgta 480
tcatcgaatt aaaccacata atccaccgct tgtgcgggcc cccgtcaatt cctttgagtt 540
tcaaacttgc gttcgtactc cccaggtggc taacttatca ctttcgctta gtctctgaaa 600
tttacatccc aaaaacgagt tagcatcgtt tacggcgtgg actaccaggg tatctaatcc 660
tgttcgctcc ccacgctttc gtccatcagc gtcagttgtt gcttagtaac ctgccttcgc 720
aattggtgtt ctaagtaata tctatgcatt tcaccgctac actacttatt ccagctactt 780
caacaacact caagacatgc agtatcaatg gcagtttcac agttaagctg tgagatttca 840
ccactgactt acacatcagc ctacggaccc tttaaaccca ataaatccgg ataacgcttg 900
caccctccgt attaccgcgg ctgctggcac ggagttagcc gggtgcttat tcgtatagta 960
ccttcagcta ctctcacgag agtaggttta tccctannac aaaaagaag 1009

Claims (10)

1. A feather high-efficiency degradation strain is classified and named as Flavobacterium lutescens (F.lutescens) of ChryseobacteriumFlavobacterium tirrenicum) X-Y4, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019286, preservation date of 2019, 4 months and 23 days.
2. Use of the strain of claim 1 for degrading feathers to produce proline.
3. The application of claim 2, wherein the strain is activated, coated on a feather solid culture medium, and cultured for 16-24 h at 30-40 ℃; selecting strains on the feather solid culture medium, purifying and streaking the strains on a fresh feather solid culture medium, and culturing the strains at the temperature of 30-40 ℃ for 16-24 h to form single colonies; and (3) selecting a single colony on the feather solid culture medium, inoculating the single colony in an LB liquid culture medium, culturing for 16-24 h at 25-40 ℃, then inoculating the single colony in the feather liquid culture medium, and culturing for 60-80 h at 25-40 ℃ to obtain the proline-containing fermentation liquid.
4. The use of claim 3, wherein the feather solid medium is formulated as: 10g of feather, 1g of sodium chloride, 1g of dipotassium hydrogen phosphate, 0.4g of monopotassium phosphate, 20g of agar powder and 1000ml of water.
5. The use according to claim 3, wherein the LB liquid medium is formulated as: 10.0g/L of peptone, 5.0g/L of yeast powder, 10.0g/L of sodium chloride and 1000ml of water.
6. The use of claim 3, wherein the feather liquid medium is formulated as: 10g of feather, 1g of sodium chloride, 1g of dipotassium phosphate, 0.4g of monopotassium phosphate and 1000ml of water.
7. Use according to claim 3, wherein the proline concentration in the final fermentation broth is above 1.0g/L and the mass percentage of proline in the degraded amino acid product is above 92%.
8. The use of the strain of claim 1 for degrading feathers to produce fertilizer.
9. The application of claim 8, wherein the strain is activated, coated on a feather solid culture medium, and cultured for 16-24 h at 30-40 ℃; selecting strains on the feather solid culture medium, purifying and streaking the strains on a fresh feather solid culture medium, and culturing the strains at the temperature of 30-40 ℃ for 16-24 h to form single colonies; and (2) selecting a single colony on the feather solid culture medium, inoculating the single colony in an LB liquid culture medium, culturing for 16-24 h at 25-40 ℃, inoculating the single colony in the feather liquid culture medium, culturing for 60-80 h at 25-40 ℃ to obtain a fermentation liquor containing proline, and centrifuging the fermentation liquor to obtain a supernatant serving as a liquid fertilizer.
10. The use according to claim 9, characterized in that the specific application method is: root irrigation is carried out for 1 time every 6-8 days, and the application amount is 0.2-0.6L/m2And 3-5 times, and the application concentration is 5-15 mmol/L calculated by the concentration of proline.
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