CN110305819A - One plant of feather efficient degrading bacterial strain and its application - Google Patents
One plant of feather efficient degrading bacterial strain and its application Download PDFInfo
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- CN110305819A CN110305819A CN201910757813.8A CN201910757813A CN110305819A CN 110305819 A CN110305819 A CN 110305819A CN 201910757813 A CN201910757813 A CN 201910757813A CN 110305819 A CN110305819 A CN 110305819A
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- 210000003746 feather Anatomy 0.000 title claims abstract description 156
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 34
- 230000000593 degrading effect Effects 0.000 title claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 47
- 239000003337 fertilizer Substances 0.000 claims abstract description 46
- 230000015556 catabolic process Effects 0.000 claims abstract description 42
- 238000006731 degradation reaction Methods 0.000 claims abstract description 42
- 238000000855 fermentation Methods 0.000 claims abstract description 40
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 37
- 241000894006 Bacteria Species 0.000 claims abstract description 18
- 241000589565 Flavobacterium Species 0.000 claims abstract description 12
- 241000611330 Chryseobacterium Species 0.000 claims abstract description 10
- 238000003973 irrigation Methods 0.000 claims abstract description 8
- 230000002262 irrigation Effects 0.000 claims abstract description 8
- 239000002609 medium Substances 0.000 claims description 54
- 239000007787 solid Substances 0.000 claims description 33
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- 238000000034 method Methods 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000012530 fluid Substances 0.000 claims description 18
- 235000015097 nutrients Nutrition 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 13
- 150000001413 amino acids Chemical class 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 abstract description 31
- 150000003839 salts Chemical class 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 11
- 230000008635 plant growth Effects 0.000 abstract description 8
- 239000002699 waste material Substances 0.000 abstract description 7
- 239000002028 Biomass Substances 0.000 abstract description 6
- 230000035882 stress Effects 0.000 abstract description 6
- 230000006518 acidic stress Effects 0.000 abstract description 2
- 238000009374 poultry farming Methods 0.000 abstract description 2
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 230000000050 nutritive effect Effects 0.000 abstract 1
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- 238000011069 regeneration method Methods 0.000 abstract 1
- 239000002689 soil Substances 0.000 description 46
- 244000221633 Brassica rapa subsp chinensis Species 0.000 description 31
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 22
- 239000002253 acid Substances 0.000 description 15
- 238000004519 manufacturing process Methods 0.000 description 14
- 239000002054 inoculum Substances 0.000 description 13
- 241000196324 Embryophyta Species 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 238000012360 testing method Methods 0.000 description 8
- 102000011782 Keratins Human genes 0.000 description 7
- 108010076876 Keratins Proteins 0.000 description 7
- 239000000654 additive Substances 0.000 description 7
- 230000000996 additive effect Effects 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 5
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 5
- 239000007836 KH2PO4 Substances 0.000 description 5
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- 239000003643 water by type Substances 0.000 description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
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- 229910052700 potassium Inorganic materials 0.000 description 4
- 239000011591 potassium Substances 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 230000003204 osmotic effect Effects 0.000 description 3
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- 238000011160 research Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101710200191 Feather keratin Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
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- 235000015177 dried meat Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 244000005700 microbiome Species 0.000 description 2
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- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
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- 239000005446 dissolved organic matter Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010059345 keratinase Proteins 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 238000012803 optimization experiment Methods 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F1/00—Fertilisers made from animal corpses, or parts thereof
- C05F1/005—Fertilisers made from animal corpses, or parts thereof from meat-wastes or from other wastes of animal origin, e.g. skins, hair, hoofs, feathers, blood
-
- 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
-
- 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
-
- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/24—Proline; Hydroxyproline; Histidine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/20—Flavobacterium
-
- Y—GENERAL 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
-
- Y—GENERAL 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention discloses one plant of feather efficient degrading bacterial strains, its classification naming is Chryseobacterium sp (Flavobacterium tirrenicum), bacterial strain X-Y4, it has been preserved in China typical culture collection center, deposit number is CCTCC NO:M 2019286, and the deposit date is on April 23rd, 2019.The invention also discloses above-mentioned Chryseobacterium sps to prepare the application in proline in degradation of feather.The bacterial strain of institute's breeding of the present invention can effectively solve the regeneration of waste feathers, can be used as the liquid fertilizer of field irrigation, realize the comprehensive utilization of biomass resource, while reduce pollution of the poultry farming to environment.Since the bacterium degradation capability is stronger, fermentation period is short, and proline content is 92% or more in fermentation liquid.The fermentation liquid of Pro-rich can be prepared into Liquid Fertilizer, has the effects that promote plant growth, alleviate salt stress and alleviate acid stress, enhances the nutritive value and utilization rate as fertilizer.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to one plant of feather degradation bacteria and its application.
Background technique
A large amount of consumption of animal generate many feather wastes, and traditional treatment method predominantly fills, burns and produce gas, right
Environment causes very big harm.The research for preparing animal feed using poultry feather is more early, has been achieved with the application hair of certain scale
Exhibition, but 90% keratin is not efficiently utilized in feather, causes the waste of available resources.Keratin is as the main of feather
Ingredient has been applied to cosmetic industry, or the component as composite material, fibre is utilized.The keratin in China provides
It is abundant for source electrode, and especially in modern agriculture, large-scale poultry farming produces a large amount of keratin waste, and mesoptile is useless
Gurry yield is most, and annual output is up to more than 70 ten thousand tons.According to surveying and determination, the crude protein content of feather keratin is about 80% or more, amino
Acid content also contains macroelement, microelement, vitamin and some unknown growth factor 70% or more.Therefore feather
Keratin waste has very extensive purposes by processing appropriate.
Keratin becomes complicated tridimensional network, with good stability and indissoluble because of the crosslinking of a large amount of disulfide bond
Property, conventional method is difficult to be hydrolyzed, and thus causes the research boom of Feather degradation method.Common approach has mechanical degradation at present
Method, chemical degradation method and biotechnology method.Although physics, chemical method research are more early, exist and destroy amino acid structure, product
The disadvantages of single and product stability is poor.Relative to conventional physical, chemical method, waste feathers angle is handled with biotechnology method
Albumen, has that reaction condition is mild, product recoveries are high, environmental-friendly, can be energy saving many advantages, such as.Biotechnology method master
It to include enzymatic isolation method and microbial degradation method.Microbial degradation method is the spy for utilizing microorganism to generate keratinase degradation keratin
Property, keratin is thoroughly degraded to polypeptide and amino acid, provides possibility for the deep utilization of feather.And wherein proline is one
Kind dissolved organic matter, is adjusted the osmotic potential in cytoplasm, plays an important role in the defense mechanism by stress cell.Dried meat ammonia
Acid mainly by keeping osmotic equilibrium, stablizing the subcellular structures such as film and albumen and Scavenger of ROS, resist by protection cell
Salt stress adverse effect.Proline degradation can provide carbon source, nitrogen source and energy.Under environment-stress, applying proline outside can be mentioned
The resistance of high plant.
The report that feather can be efficiently degraded to the bacterial strain of high-purity proline is had no in the prior art.
Summary of the invention
The present invention provide it is a kind of can efficient degradation waste feathers bacterial strain, which can be degraded to feather in the dried meat of high-purity
Propylhomoserin.
The present invention also technical problems to be solved are to provide above-mentioned feather and prepare application in proline in fermentation.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
One plant of feather efficient degrading bacterial strain, classification naming are Chryseobacterium sp (Flavobacterium tirrenicum),
Bacterial strain X-Y4, has been preserved in China typical culture collection center, and deposit number is CCTCC NO:M 2019286, preservation day
Phase is on April 23rd, 2019, and preservation address is Wuhan, China Wuhan University.
The Flavobacterium tirrenicum X-Y4 of efficient feather degradation of the present invention, has following biology
Learn feature: morphological feature: thallus is rod-shaped, does not produce spore, without motion, and Gram's staining is negative, in nutrient agar culture
Yellow colonies (as shown in Figure 1) is formed on base.
The process of screening, includes the following steps:
A. the preparation of Selective agar medium
B. the primary dcreening operation of feather degradation bacteria
C. the secondary screening of feather degradation bacteria
The step a may further include: preparing feather solid medium, prepares feather fluid nutrient medium.
The step b may further include: in the dove factory pedotheque after air-drying, weighing 5g soil and is added to 50ml
Sterile water in enrichment culture, 30 DEG C of condition of culture, 160r/min cultivate 12h；Take enrichment culture liquid by 10-1、10-2、 10-3、
10-4、10-5、10-6、10-7、10-8Gradient dilution is coated on feather solid medium, purifying scribing line training when growing single colonie
It supports 5 times, by the secondary screening of feather fluid nutrient medium, to obtain efficient feather degradation bacteria.
The step c may further include: by the strain inoculated screened in 100ml feather fluid nutrient medium, 30
DEG C, 160r/min cultivate 3d respectively, measure bacterial strain production proline ability.
Bacterial strain identification method:
16sRNA sequence analysis: PCR amplification uses bacterium 16sRNA universal primer:
27F5’-AGAGTTTGATCMTGGCTCAG-3’
1492R5’-GGTTACCTTGTTACGACTT-3’
PCR reaction system (50 μ L): 10 × amplification buffer (5 μ L), genomic DNA (1 μ L), dNTP (2 μ L), primers F
(2 μ L), primer R (2 μ L), Taq archaeal dna polymerase (0.25 μ L), deionized water (37.75 μ L).Reaction condition: 95 DEG C of initial denaturations
10min, 95 DEG C of deformations 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 25 recycle, and 72 DEG C sufficiently extend 10min.
16S rRNA major part sequence is measured, as shown in SEQ ID No:1.
Strain X-Y4 has the following properties:
1, colonial morphology feature:
On LB solid medium 30 DEG C culture 1~2 day after microscope observe vegetative cell be it is unicellular, it is rod-shaped.?
30 DEG C of culture 12h thallus can be with raised growth in above-mentioned culture medium.Bacterium colony is in golden yellow, and round, surface is smooth, neat in edge,
It is sticky, intermediate projections.
2, physio-biochemical characteristics:
The physiological and biochemical property of 1. bacterial strain of table
3,16S rDNA sequence is analyzed
27F 5’-AGAGTTTGATCMTGGCTCAG-3’
1492R 5’-GGTTACCTTGTTACGACTT-3’
PCR reaction system (50 μ L): 10 × amplification buffer (5 μ L), genomic DNA (1 μ L), dNTP (2 μ L), primers F
(2 μ L), primer R (2 μ L), Taq archaeal dna polymerase (0.25 μ L), deionized water (37.75 μ L).Reaction condition: 95 DEG C of initial denaturations
10min, 95 DEG C of deformations 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 25 recycle, and 72 DEG C sufficiently extend 10min.
16S rDNA major part sequence is measured, as shown in SEQ ID No:1.The BLAST of the column website NCBI will be sequenced
The comparison of base sequence is carried out, the phylogenetic tree based on 16S rRNA complete sequence is constructed.The result shows that: bacterial strain and golden yellow bar
It is homologous that bacterium reaches 98%.So assert that the present invention uses Chryseobacterium sp (Flavobacterium tirrenicum), specifically
Position Chryseobacterium sp Flavobacterium tirrenicum X-Y4.
Above-mentioned bacterial strains prepare the application in proline also within protection scope of the present invention in degradation of feather.
The method that above-mentioned bacterial strains degradation of feather prepares fertilizer is to activate bacterial strain, is coated on feather solid medium,
30 DEG C~40 DEG C cultures 16~for 24 hours (preferably 18h)；Strain on picking feather solid medium is trained in fresh feather solid
It supports and purifies scribing line on base, 30 DEG C~40 DEG C cultures 16~for 24 hours (preferably 18h) form single colonie；On picking feather solid medium
Single colonie, be inoculated in LB liquid medium, 25 DEG C~40 DEG C cultures 16~(preferably culture is to OD for 24 hours600=0.6) it, then connects
Kind is in feather fluid nutrient medium, and inoculum concentration 2%v/v, 25 DEG C~40 DEG C (preferably 35 DEG C) are cultivated 60~80 hours (preferably
72h), the fermentation liquid containing proline is obtained.
Wherein, the feather solid culture based formulas is preferably: feather 10g, sodium chloride 1g, dipotassium hydrogen phosphate 1g, phosphorus
Acid dihydride potassium 0.4g, agar powder 20g, water 1000ml.
Wherein, the formula of the LB liquid medium is preferred are as follows: peptone 10.0g/L, yeast powder 5.0g/L, sodium chloride
10.0g/L, water 1000ml.
Wherein, the feather Liquid Culture based formulas is preferably: feather 10g, sodium chloride 1g, dipotassium hydrogen phosphate 1g, phosphorus
Acid dihydride potassium 0.4g, water 1000ml.
Wherein, in final fermentation liquid, concentration of proline is 1.0g/L or more, and proline accounts in the amino acid product of degradation
Mass percent is 92% or more.
Available amino acid rich in the tunning of bacterial strain of the present invention, degradation efficiency is high, fermentation period
It is short, the loss of Most amino-acids is avoided, the utilization rate of feather catabolite is improved, proline content exists in fermentation liquid
92% or more.Proline is plant inner product under the environment stresses such as arid, high temperature, with high salt, frost, ultraviolet light and heavy metal
Tired main osmotic adjustment.And the fermentation liquid of Pro-rich can be prepared into Liquid Fertilizer, has and promotes plant growth, alleviates
The effects of salt stress and alleviation acid stress.
Above-mentioned bacterial strains prepare the application in fertilizer also within protection scope of the present invention in degradation of feather.
The method that above-mentioned bacterial strains degradation of feather prepares fertilizer is to activate bacterial strain, is coated on feather solid medium,
30 DEG C~40 DEG C cultures 16~for 24 hours (preferably 18h)；Strain on picking feather solid medium is trained in fresh feather solid
It supports and purifies scribing line on base, 30 DEG C~40 DEG C cultures 16~for 24 hours (preferably 18h) form single colonie；On picking feather solid medium
Single colonie, be inoculated in LB liquid medium, 25 DEG C~40 DEG C cultures 16~(preferably culture is to OD for 24 hours600=0.6) it, then connects
Kind is in feather fluid nutrient medium, and inoculum concentration 2%v/v, 25 DEG C~40 DEG C (preferably 35 DEG C) are cultivated 60~80 hours (preferably
72h), the fermentation liquid containing proline is obtained, using fermentation liquid centrifuging and taking supernatant as liquid fertilizer.
Obtained fermentation liquid is preferably separated by solid-liquid separation removal thallus, retains clear liquid as liquid fertilizer.
Wherein, the feather solid culture based formulas is preferably: feather 10g, sodium chloride 1g, dipotassium hydrogen phosphate 1g, phosphorus
Acid dihydride potassium 0.4g, agar powder 20g, water 1000ml.
Wherein, the optimization formula of the LB liquid medium are as follows: peptone 10.0g/L, yeast powder 5.0g/L, sodium chloride
10.0g/L, water 1000ml.
Wherein, the feather Liquid Culture based formulas is preferably: feather 10g, sodium chloride 1g, dipotassium hydrogen phosphate 1g, phosphorus
Acid dihydride potassium 0.4g, water 1000ml.
The manufactured specific method of administration of fertilizer are as follows: root irrigation, every 6~8d are carried out 1 time, and amount of application is 0.2~0.6L/
m2, 3~5 times, application concentration is calculated as 5~15mmol/L with concentration of proline.
The stress that above-mentioned fertilizer can promote plant growth, alleviate salt and acid in soil.
It is had the advantage that the utility model has the advantages that efficient feather of the invention degrades and its prepares feather catabolite
The present invention filters out the bacterium Flavobacterium tirrenicum X- with degradation of feather ability from soil
Y4, and feather catabolite is obtained using feather degradation bacteria Flavobacterium tirrenicum X-Y4 as strain fermentation, it should
Containing the proline of high-purity in feather catabolite, amino-acid liquid fertilizer may be used as, alleviate salt stress effect and alleviate acid
Property soil can effectively improve the yield of pakchoi, and the preparation side of Liquid Fertilizer provided by the invention to the coercion of plant
Method, simple production process, high production efficiency are able to achieve industrialized production, have good economic benefits and social effect.
Detailed description of the invention
Fig. 1 is the colonial morphology on LB plate of X-Y4.
Fig. 2 is the Gram's staining of X-Y4.
Fig. 3 is the 16SrRNA sequential system evolutionary analysis example schematic of X-Y4.
Fig. 4 is histogram of the feather additive amount to soluble protein concentration.
Fig. 5 is histogram of the shaking speed to soluble protein concentration.
Fig. 6 is histogram of the initial pH to soluble protein concentration.
Fig. 7 is histogram of the inoculum concentration to soluble protein concentration.
Fig. 8 is histogram of the temperature to soluble protein concentration.
Fig. 9 is degradation effect figure of the XY-4 fermented and cultured 72h to feather.
Figure 10 is the line chart of feather degradation situation in 72 hours.
Specific embodiment
Form is described in further detail above content of the invention again by the following examples, but should not manage this
For solution for the scope of the above subject matter of the present invention is limited to the following embodiments, all technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Embodiment 1: the screening of Chryseobacterium sp X-Y4
(1) sample source: the feeding dove head phase stacks the place soil sample for the feather that rotted.
(2) bacterial strain screening:
The process of screening, includes the following steps:
A. the preparation of Selective agar medium
B. the primary dcreening operation of feather degradation bacteria
C. the secondary screening of feather degradation bacteria
The step a may further include: preparing feather solid medium, prepares feather fluid nutrient medium.
The step b may further include: in the dove factory pedotheque after air-drying, weighing 5g soil and is added to 50ml
Sterile water in enrichment culture, 30 DEG C of condition of culture, 160r/min cultivate 12h；Take enrichment culture liquid by 10-1、10-2、 10-3、
10-4、10-5、10-6、10-7、10-8Gradient dilution is coated on feather solid medium, purifying scribing line training when growing single colonie
It supports 5 times, by the primary dcreening operation of feather fluid nutrient medium, to obtain efficient feather degradation bacteria.
The step c may further include: by the strain inoculated screened in 100ml feather fluid nutrient medium, 30
DEG C, 160r/min cultivate 3d respectively, measure the content (being shown in Table 2) of proline in fermentation liquid, it is highest to filter out proline yield
Strain X-Y4.
Proline content in 2. different strains fermentation liquid of table
(3) colony morphological observation of X-Y4 bacterial strain
By the X-Y4 bacterial strain screened on feather solid medium 30 DEG C culture 1 day after carry out Gram's staining, pass through
Microscope observe vegetative cell be it is unicellular, it is rod-shaped, as shown in Figure 2.30 DEG C of culture 12h thallus can in above-mentioned culture medium
With raised growth.Bacterium colony is in golden yellow, and round, surface is smooth, neat in edge, sticky, intermediate projections, as shown in Figure 2.
Embodiment 2: strain idenfication.
16sRNA sequence analysis: PCR amplification uses bacterium 16sRNA universal primer:
27F5’-AGAGTTTGATCMTGGCTCAG-3’
1492R5’-GGTTACCTTGTTACGACTT-3’
DNTP (2 μ L), primers F (2 μ L), primer R (2 μ L), Taq archaeal dna polymerase (0.25 μ L), deionized water (37.75 μ
L).Reaction condition: 95 DEG C of initial denaturations 10min, 95 DEG C of deformations 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 25 recycle, and 72
DEG C sufficiently extend 10min.
16S rDNA major part sequence is measured, as shown in SEQ ID No:1.The BLAST of the column website NCBI will be sequenced
The comparison of base sequence is carried out, the phylogenetic tree based on 16S rRNA complete sequence is constructed.The result shows that: bacterial strain and golden yellow bar
It is homologous that bacterium reaches 98%.So assert that the present invention uses Chryseobacterium sp (Flavobacterium tirrenicum), specifically
Position Chryseobacterium sp Flavobacterium tirrenicum X-Y4.
Embodiment 3: the training systern of feather degrading bacteria
(1) bacterial strain: the bacterial strain Flavobacterium tirrenicum X-Y4 that embodiment 1 filters out
(2) method and step:
Feather additive amount, shaking speed, initial pH, inoculum concentration and the fermentation temperature pair of bacterial strain are investigated using single factor experiment
The influence of soluble protein yield.
A. the optimization test of feather additive amount
Fermentation condition be feather fluid nutrient medium fill liquid 100mL/250mL, inoculum concentration 2% (v/v), 30 DEG C, 160r/min,
PH=7.0, shake flask fermentation 72h, feather amount of filling are respectively 0.5,1.0g, 1.5g, 2.0g, 2.5g and 3.0g.
B. the optimization experiment of shaking speed
Feather addition measures the optimal result that above-mentioned test determines, other fermentation conditions are that feather fluid nutrient medium fills liquid
100mL/250mL, inoculum concentration 2% (v/v), 30 DEG C, pH=7.0, shake flask fermentation 72h, shaking speed be respectively 140r/min,
160r/min, 180r/min, 200r/min, 220r/min and 240r/min investigate shaking speed and produce soluble protein to strain
Influence.
C. the optimization test of initial pH
The optimal result that feather additive amount and shaking speed determine, other fermentation conditions are that feather fluid nutrient medium fills liquid
100mL/250mL, inoculum concentration 2% (v/v), 30 DEG C, shake flask fermentation 72h, by pre-stage test, the first initial pH difference of successive step
Influence of the pH to bacterial strain soluble protein is investigated for 8.0,8.5,9.0,9.5,10.0.
D. the optimization test of inoculum concentration
The optimal result that feather additive amount, shaking speed and initial pH take above-mentioned test to determine, other fermentation conditions are plumage
Hair fluid nutrient medium fills liquid 100mL/250mL, 30 DEG C, shake flask fermentation 72h, and inoculum concentration is respectively 1%, 2%, 3%, 4% and
5%, investigate the influence that shaking speed produces soluble protein to strain.
E. the optimization test of fermentation temperature
Feather additive amount, shaking speed, initial pH and inoculation measure the optimal result that above-mentioned test determines, other fermentation items
Part is that feather fluid nutrient medium fills liquid 100mL/250mL, shake flask fermentation 72h, respectively at 25 DEG C, 30 DEG C, 35 DEG C, 37 DEG C and 45
Soluble protein is measured after carrying out fermented and cultured in DEG C shaking table, investigates the influence that temperature produces soluble protein to strain.
As can be seen from Figure 4,122.35 ± 3.53 μ g/ml of soluble protein content highest when the feather amount of filling out is 1g；It can from Fig. 5
Know, 121.32 ± 20.76 μ g/ml of soluble protein content highest when revolving speed is 200r/min；As can be seen from Figure 6, initial pH is
162.71 ± 35.51 μ g/ml of soluble protein content highest when 9.0；As can be seen from Figure 7, soluble protein contains when inoculum concentration is 2%
Measure 168.56 ± 7.82 μ g/ml of highest；As it can be observed in the picture that 173.27 ± 2.95 μ of soluble protein content highest when temperature is 35 DEG C
g/ml.In conclusion fermentation optimal conditions are as follows: feather amount of filling is 1g, revolving speed 200r/min, initial pH are 9.0, inoculum concentration
It is 35 DEG C for 2% and temperature, highest soluble protein content is 173.27 ± 2.95 μ g/ml.
Embodiment 4: feather degradation experiment
X-Y4 is accessed into feather fluid nutrient medium, Shake flask medium liquid amount 100mL/ with the inoculum concentration of 2% (v/v)
250mL, pH=9.0,1g/L feather additive amount, 2% inoculum concentration, 35 DEG C, 200r/min shake flask fermentation 72h.Fermentation observation feather
Signs of degradation, since culture medium is without the carbon nitrogen source in addition to feather, the transparent clarification shape of culture medium.When fermentation 12h or so, feather master
Tiny lint on stalk falls off, and culture medium is gradually muddy；When fermentation proceeds to 48h, culture medium is muddy, opaque, the big portion of feather
Divide and be degraded, only the main stalk of remaining part point；After 72h, the main stalk of culture medium mesoptile is also degraded.As shown in Fig. 9, after 72h degradation and not
Degradation is compared, and solution becomes cloudy, and in golden yellow, is existed substantially without bulky grain feather in solution, feather is substantially completely degraded.Such as
Shown in Figure 10, degradation rate can reach 95.72% within 72 hours.
The product of embodiment 5:X-Y4 degradation dove hair
- 20 DEG C of feather degradation bacteria activation, the coating of Yu Yumao solid medium, 30 DEG C of culture 18h will be stored in；Picking feather
Strain on solid medium purifies scribing line, 30 DEG C of culture 18h on fresh feather solid medium；Picking feather solid
Single colonie on culture medium, is inoculated in LB liquid medium, 35 DEG C of culture 18h, 35 DEG C of culture medium in feather fluid nutrient medium
Culture 72 hours, obtains fermentation liquid.Fermentation liquid is centrifuged 15 minutes under the conditions of 4 DEG C, 6000r/min, supernatant is taken, by supernatant
It is stored in 4 DEG C of refrigerators.Herein experiment mesoptile be dove hair.
Supernatant is feather catabolite, can be used as amino-acid liquid fertilizer, wherein proline content accounting is up to
92.58%.
The content of amino acid in the product of the degradation dove hair of table 3.
The product of embodiment 6:X-Y4 degradation chicken feather
- 20 DEG C of feather degradation bacteria activation, the coating of Yu Yumao solid medium, 30 DEG C of culture 18h will be stored in；Picking feather
Strain on solid medium purifies scribing line, 30 DEG C of culture 18h on fresh feather solid medium；Picking feather solid
Single colonie on culture medium, is inoculated in LB liquid medium, 35 DEG C of culture 18h, 35 DEG C of culture medium in feather fluid nutrient medium
Culture 72 hours, obtains fermentation liquid.Fermentation liquid is centrifuged 15 minutes under the conditions of 4 DEG C, 6000r/min, supernatant is taken, by supernatant
It is stored in 4 DEG C of refrigerators.Herein experiment mesoptile be chicken feather.
The content of amino acid in the product of the degradation chicken feather of table 4.
Embodiment 7: feather catabolite difference applies influence of the concentration to crop yield
Flowerpot bore 140mm, the high 115mm used is tested, every basin fills soil 650g, and rate of fertilizer is 0.571 gkg- 1NH4NO3, 0.439gkg-1KH2PO4, 0.141gkg-1KCl mixes fertilizer and soil thoroughly dress basin, and being put into suitable water makes soil
Earth soaks completely.Seed is soaked in pure water overnight (4 DEG C), selects full consistent seed in plastic tub, every basin sowing
About 5, dispersion distance is uniform, and culture is to germinateing in greenhouse, and thinning is to 2 after germination, with different volumes feather catabolite
Solution carries out root irrigation, concentration of proline 5mmol/L, and every 7d is carried out 1 time, each dosage 0.2L/m2, concentration of proline
Respectively 10mmol/L and 5mmol/L 3 times in total, during which waters daily, so that moisture is maintained about 75% field with weight minusing
Between water-holding capacity, co-culture 30d.Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, in which:
Control group: normal soil+fertilizer
Experimental group 1: normal soil+fertilizer+0.2L/m2Feather catabolite (proline of 10mmol/L)
Experimental group 2: normal soil+fertilizer+0.2L/m2Feather catabolite (proline of 5mmol/L)
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
5. feather catabolite different administration concentration of table is to pakchoi characteristic and yield effect
As shown in Table 5, feather catabolite can improve pakchoi plant height and fresh weight in various degree, add feather catabolite
(proline of 10mmol/L) and be not added feather catabolite comparison volume increase 21.10%, add feather catabolite (5mmol/L's
Proline) it still can increase production 7.07%, illustrate that feather catabolite has facilitation to the growth of pakchoi, in certain model
Interior its higher effect of application concentration is enclosed to be more obvious.
Embodiment 8: feather catabolite difference applies influence of the number to crop yield
Flowerpot bore 140mm, the high 115mm used is tested, every basin fills soil 650g, and rate of fertilizer is 0.571 gkg- 1NH4NO3, 0.439gkg-1KH2PO4, 0.141gkg-1KCl mixes fertilizer and soil thoroughly dress basin, and being put into suitable water makes soil
Earth soaks completely.Seed is soaked in pure water overnight (4 DEG C), selects full consistent seed in plastic tub, every basin sowing
About 5, dispersion distance is uniform, and culture is to germinateing in greenhouse, and thinning is to 2 after germination, with various concentration feather catabolite
Solution carries out root irrigation, and every 4d, 5d, 7d are carried out 1 time, each dosage 0.2L/m2, concentration of proline is 5mmol/L when application.
It 5,4,3 times in total, during which waters daily, so that moisture is maintained about 75% field capacity with weight minusing, co-culture 30d.
Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, in which:
Control group: normal soil+fertilizer
Experimental group 1: normal soil+fertilizer+0.2L/m2Feather catabolite (primary every 4d fertilising, to apply fertilizer 5 times altogether)
Experimental group 2: normal soil+fertilizer+0.2L/m2Feather catabolite (primary every 5d fertilising, to apply fertilizer 4 times altogether)
Experimental group 3: normal soil+fertilizer+0.2L/m2Feather catabolite (primary every 7d fertilising, to apply fertilizer 3 times altogether)
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
6. feather catabolite difference of table applies number to pakchoi characteristic and yield effect
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with the 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 shown in Table 6, the feather catabolite for applying different numbers can improve pakchoi plant height and fresh weight in various degree,
Every 4d fertilising once be not added feather catabolite comparison volume increase 24.36%, every 5d fertilising can once increase production 17.85%
And once can illustrate that feather catabolite has facilitation to the growth of pakchoi every 7d fertilising with getting fat 16.12%,
Apply the more gains of number in a certain range to be more obvious.
Embodiment 9: influence of the feather catabolite difference applied amount to crop yield.
Flowerpot bore 140mm, the high 115mm used is tested, every basin fills soil 650g, and rate of fertilizer is 0.571 gkg- 1NH4NO3, 0.439gkg-1KH2PO4, 0.141gkg-1KCl mixes fertilizer and soil thoroughly dress basin, and being put into suitable water makes soil
Earth soaks completely.Seed is soaked in pure water overnight (4 DEG C), selects full consistent seed in plastic tub, every basin sowing
About 5, dispersion distance is uniform, and culture is to germinateing in greenhouse, and thinning is to 2 after germination, with various concentration feather catabolite
Solution carries out root irrigation, and every 7d is carried out 1 time, each dosage 0.2L/m2、 0.4L/m2And 0.6L/m2, proline is dense when application
Degree is 5mmol/L.It 3 times in total, during which waters daily, so that moisture is maintained about 75% field capacity with weight minusing, altogether
Cultivate 30d.Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, in which:
Control group 1: normal soil+fertilizer
Experimental group 1: normal soil+fertilizer+0.2L/m2Feather catabolite
Experimental group 2: normal soil+fertilizer+0.4L/m2Feather catabolite
Experimental group 3: normal soil+fertilizer+0.6L/m2Feather catabolite
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
7. feather catabolite difference applied amount of table is to pakchoi characteristic and yield effect
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with the 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% |
| Experimental group 3 | 21.23±0.19 | 31.11±0.28 | 25.49% |
As shown in Table 7, apply different applied amounts feather catabolite can improve in various degree pakchoi plant height with it is fresh
Weight, applies 0.2L/m2Feather catabolite increases production 19.36% with the comparison of feather catabolite is not added, and applies 0.4L/m2Feather degradation produces
Object can increase production 24.04% and apply 0.6L/m2Feather catabolite can illustrate feather catabolite pair with getting fat 25.49%
The growth of pakchoi has facilitation, and applied amount is got over multiple-effect fruit and is more obvious in a certain range.
Embodiment 10: feather catabolite can alleviate salt affected soil to the coercion of pakchoi.
Flowerpot bore 140mm, the high 115mm used is tested, every basin fills soil 650g, and rate of fertilizer is 0.571 gkg- 1NH4NO3, 0.439gkg-1KH2PO4, 0.141gkg-1KCl mixes fertilizer and salt affected soil thoroughly dress basin, is put into suitable water
Soak soil completely.Seed is soaked in pure water overnight (4 DEG C), selects full consistent seed in plastic tub, every basin
Sowing about 5, dispersion distance is uniform, and culture is to germinateing in greenhouse, and thinning is degraded to 2 with different volumes feather after germination
Reaction mixture carries out root irrigation, and every 7d is carried out 1 time, each dosage 0.2L/m2Or 0.4L/m2, concentration of proline is when application
15mmol/L.It 3 times in total, during which waters daily, so that moisture is maintained about 75% field capacity with weight minusing, co-culture
30d.Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, in which:
Control group: salt affected soil+fertilizer
Experimental group 1: salt affected soil+fertilizer+0.2L/m2Feather catabolite
Experimental group 2: salt affected soil+fertilizer+0.4L/m2Feather catabolite
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
8 feather catabolite of table is to salt affected soil pakchoi characteristic and yield effect
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with the 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 shown in Table 8, apply feather catabolite and significantly improve pakchoi plant height and fresh weight in salt affected soil, apply
0.2L/m2Feather catabolite increases production 24.71%, 0.4L/m with the comparison of feather catabolite is not added2Feather catabolite can increase
38.84% is produced, illustrates that feather catabolite can also play the growth-promoting functions to pakchoi in alkaline land soil.
Embodiment 11: feather catabolite can alleviate acid soil to the coercion of pakchoi.
Flowerpot bore 140mm, the high 115mm used is tested, every basin fills soil 650g, and rate of fertilizer is 0.571 gkg- 1NH4NO3, 0.439gkg-1KH2PO4, 0.141gkg-1KCl mixes fertilizer and acid soil thoroughly dress basin, is put into suitable water
Soak soil completely.Seed is soaked in pure water overnight (4 DEG C), selects full consistent seed in plastic tub, every basin
Sowing about 5, dispersion distance is uniform, and culture is to germinateing in greenhouse, and thinning is degraded to 2 with various concentration feather after germination
Reaction mixture carries out root irrigation, and every 7d is carried out 1 time, and each dosage is respectively 0.2L/m2、0.4L/m2, proline is dense when application
Degree is 5mmol/L.It 3 times in total, during which waters daily, so that moisture is maintained about 75% field capacity with weight minusing, altogether
Cultivate 30d.Using ?leaf palm-leaf fun Chinese cabbage as experimental subjects, in which:
Control group: acid soil+fertilizer
Experimental group 1: acid soil+fertilizer+0.2L/m2Feather catabolite
Experimental group 2: acid soil+fertilizer+0.4L/m2Feather catabolite
Each processing group harvests, plant is cut along root, is eluted with water, biomass is measured for the 30th day in plant growth.
9. feather catabolite of table is to acid soil pakchoi characteristic and yield effect
| Experimental group | Pakchoi plant height (cm) | Pakchoi fresh weight (g/ basin) | Increase production compared with the 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 shown in Table 9, apply feather catabolite and significantly improve pakchoi plant height and fresh weight in acid soil, apply
0.2L/m2Feather catabolite increases production 24.71% with the comparison of feather catabolite is not added, and applies 0.4L/m2Feather catabolite can be with
Volume increase 38.84%, illustrates that feather catabolite can also play the growth-promoting functions to pakchoi in acid soil.
Sequence table
<110>Nanjing University of Technology
<120>one plants of feather efficient degrading bacterial strains and its applications
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1009
<212> DNA
<213>Chryseobacterium sp (Flavobacterium tirrenicum)
<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 gcaggactta 360
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. one plant of feather efficient degrading bacterial strain, classification naming is Chryseobacterium sp (Flavobacterium tirrenicum), bacterium
Strain X-Y4, has been preserved in China typical culture collection center, and deposit number is CCTCC NO:M 2019286, preservation date
For on April 23rd, 2019.
2. bacterial strain described in claim 1 prepares the application in proline in degradation of feather.
3. application according to claim 2, which is characterized in that activate bacterial strain, be coated on feather solid medium, 30
DEG C~40 DEG C of cultures 16~for 24 hours；Strain on picking feather solid medium, purifying is drawn on fresh feather solid medium
Line, 30 DEG C~40 DEG C of cultures 16~form single colonie for 24 hours；Single colonie on picking feather solid medium, is inoculated in LB liquid
In culture medium, 25 DEG C~40 DEG C cultures 16~for 24 hours, it is inoculated in feather fluid nutrient medium, 25 DEG C~40 DEG C cultures 60~80
Hour, obtain the fermentation liquid containing proline.
4. application according to claim 3, which is characterized in that the feather solid culture based formulas are as follows: feather 10g,
Sodium chloride 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 0.4g, agar powder 20g, water 1000ml.
5. application according to claim 3, which is characterized in that the formula of the LB liquid medium are as follows: peptone
10.0g/L, yeast powder 5.0g/L, sodium chloride 10.0g/L, water 1000ml.
6. application according to claim 3, which is characterized in that the feather Liquid Culture based formulas are as follows: feather 10g,
Sodium chloride 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 0.4g, water 1000ml.
7. application according to claim 3, which is characterized in that in final fermentation liquid, concentration of proline is 1.0g/L or more,
It is 92% or more that proline, which accounts for the mass percent in the amino acid product of degradation,.
8. bacterial strain described in claim 1 prepares the application in fertilizer in degradation of feather.
9. application according to claim 8, which is characterized in that activate bacterial strain, be coated on feather solid medium, 30
DEG C~40 DEG C of cultures 16~for 24 hours；Strain on picking feather solid medium, purifying is drawn on fresh feather solid medium
Line, 30 DEG C~40 DEG C of cultures 16~form single colonie for 24 hours；Single colonie on picking feather solid medium, is inoculated in LB liquid
In culture medium, 25 DEG C~40 DEG C cultures 16~for 24 hours, it is inoculated in feather fluid nutrient medium, 25 DEG C~40 DEG C cultures 60~80
Hour, the fermentation liquid containing proline is obtained, using fermentation liquid centrifuging and taking supernatant as liquid fertilizer.
10. application according to claim 9, which is characterized in that specific method of administration are as follows: root irrigation, every 6~8d are carried out
1 time, amount of application is 0.2~0.6L/m2, 3~5 times, application concentration is calculated as 5~15mmol/L with concentration of proline.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112939700A (en) * | 2021-04-13 | 2021-06-11 | 南京工业大学 | Method for preparing bio-organic fertilizer by using kitchen garbage |
| WO2022007145A1 (en) * | 2020-07-09 | 2022-01-13 | 中国农业科学院生物技术研究所 | Gene module for efficiently degrading feather and synthesizing artificial hemeprotein, and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022007145A1 (en) * | 2020-07-09 | 2022-01-13 | 中国农业科学院生物技术研究所 | Gene module for efficiently degrading feather and synthesizing artificial hemeprotein, and application thereof |
| CN112939700A (en) * | 2021-04-13 | 2021-06-11 | 南京工业大学 | Method for preparing bio-organic fertilizer by using kitchen garbage |
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