CN108676753A - Geobacillus stearothermophilus, microbial inoculum and application of bacillus stearothermophilus in treatment of kitchen waste - Google Patents
Geobacillus stearothermophilus, microbial inoculum and application of bacillus stearothermophilus in treatment of kitchen waste Download PDFInfo
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- CN108676753A CN108676753A CN201810554604.9A CN201810554604A CN108676753A CN 108676753 A CN108676753 A CN 108676753A CN 201810554604 A CN201810554604 A CN 201810554604A CN 108676753 A CN108676753 A CN 108676753A
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- 241000193385 Geobacillus stearothermophilus Species 0.000 title claims abstract description 75
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 12
- 239000010806 kitchen waste Substances 0.000 title abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 42
- 229920002472 Starch Polymers 0.000 claims abstract description 27
- 235000019698 starch Nutrition 0.000 claims abstract description 27
- 239000008107 starch Substances 0.000 claims abstract description 27
- 239000007787 solid Substances 0.000 claims abstract description 18
- 238000002360 preparation method Methods 0.000 claims abstract description 15
- 108020004465 16S ribosomal RNA Proteins 0.000 claims abstract description 6
- 239000010813 municipal solid waste Substances 0.000 claims description 78
- 241000894006 Bacteria Species 0.000 claims description 37
- 230000001580 bacterial effect Effects 0.000 claims description 32
- 239000001963 growth medium Substances 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 26
- 239000003795 chemical substances by application Substances 0.000 claims description 25
- 238000000855 fermentation Methods 0.000 claims description 22
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- 238000010411 cooking Methods 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 239000000417 fungicide Substances 0.000 claims description 16
- 230000000855 fungicidal effect Effects 0.000 claims description 14
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 13
- 230000004913 activation Effects 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 241000726221 Gemma Species 0.000 claims description 11
- 235000015278 beef Nutrition 0.000 claims description 11
- 238000011534 incubation Methods 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 9
- 239000008272 agar Substances 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 9
- 239000002689 soil Substances 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- 210000000988 bone and bone Anatomy 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 230000018044 dehydration Effects 0.000 claims description 5
- 238000006297 dehydration reaction Methods 0.000 claims description 5
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000009423 ventilation Methods 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 4
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 4
- 230000001788 irregular Effects 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 101000659581 Geobacillus stearothermophilus Anthranilate synthase component 1 Proteins 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 230000006641 stabilisation Effects 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 2
- 238000011105 stabilization Methods 0.000 abstract 1
- 230000015556 catabolic process Effects 0.000 description 15
- 238000006731 degradation reaction Methods 0.000 description 15
- 230000017854 proteolysis Effects 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
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- 238000009264 composting Methods 0.000 description 3
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- 244000005700 microbiome Species 0.000 description 3
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- 238000004064 recycling Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 230000037303 wrinkles Effects 0.000 description 2
- BHELIUBJHYAEDK-OAIUPTLZSA-N Aspoxicillin Chemical compound C1([C@H](C(=O)N[C@@H]2C(N3[C@H](C(C)(C)S[C@@H]32)C(O)=O)=O)NC(=O)[C@H](N)CC(=O)NC)=CC=C(O)C=C1 BHELIUBJHYAEDK-OAIUPTLZSA-N 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010068052 Mosaicism Diseases 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003625 amylolytic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002541 mycostatic Substances 0.000 description 1
- 230000001557 mycostatic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
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Classifications
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- 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
- C12R2001/07—Bacillus
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
The invention discloses a thermophilic geobacillus stearothermophilus A21 with a preservation number of CCTCC NO: m2018137, the 16S rDNA sequence of which is shown in SEQ ID NO: 1 is shown. The invention also discloses a preparation method of the geobacillus stearothermophilus, a liquid microbial inoculum and a solid microbial inoculum thereof, and application of the geobacillus stearothermophilus in treatment of kitchen waste. The invention can rapidly degrade starch and protein with higher content in the kitchen waste and inhibit the generation of odor, can be used for rapidly treating the kitchen waste, and realizes the rapid biological reduction and stabilization of the kitchen waste.
Description
Technical field
The present invention relates to microorganisms technical fields, and in particular to Geobacillus stearothermophilus and microbial inoculum and application.
Background technology
In recent years, with the improvement of people ' s living standards with the continuous development of catering industry, the quantity of kitchen garbage is also in
Growth trend year by year, accounts for the nearly half of domestic waste, if cannot deal carefully with, rubbish from cooking pollutes the environment, grows mosquito
Fly causes transmission, so that endangering human health.Kitchen garbage is mainly derived from leftover, wherein moisture content up to 70%,
Therefore be not suitable for handling by the way of burning, and sanitary landfills can occupy a large amount of land resource, therefore kitchen garbage is handled
Problem becomes to be on the rise with the continuous increase of kitchen garbage quantity.Biological treatment mode has become kitchen garbage processing
Preferred manner, but the characteristics of kitchen garbage also usually contains high salinity, high protein, Hi CHO and high fat content, adopt
Stink is easy tod produce when with biological treatment mode.Therefore most importantly the suitable kitchen garbage of separation screening handles microbial bacteria
Strain, can carry out efficient kitchen garbage degradation, and the organic matter degradation in kitchen garbage is quickly become water and carbon dioxide, realize
The minimizing of kitchen garbage and innoxious reduces kitchen garbage processing and at the same time inhibiting to generate the growth of the microorganism of stink
The generation of stink in the process.
Currently, biological treatment is using biological fast degradation technology as core, but need the processes such as separate waste collection.And it is biological
Mainly there are two direction, a biological treatment energy researchs for processing；Such as Jiangsu Clean Environment Technology Co., Ltd. (Zhang Longsheng
Deng 2009) that studies recycles the oily conversion biodiesel in kitchen garbage, the Sunkeehan researchs of Korean science institute
Group (2004) converts garbage into ammonia processed, and the Xiao Benyi researcher (2004) of the China side Chinese Academy of Sciences has invented diphasic anaerobic hair
Ferment technology, improves the recycling of Organic biological mass-energy in kitchen garbage, and foreign countries also have recycling kitchen garbage fermentation production glass ingeniously sour
With the report of diacid steel (Doyle et al, 1982), these research work provide very for the energy research of kitchen garbage
Mostly beneficial exploration, but because the application of biological new energy also has many bottleneck sex chromosome mosaicisms, therefore these researchs are more still at present
So rest on scientific research level.And another direction is biofermentation recycling treatment, production at present is above commonly used to get the nod
Be exactly kitchen garbage Composting application, the abundant nutriment of kitchen garbage can meet microorganism growth nutritional need,
It is highly suitable for being used as composting material, composting technology occurs as soon as very early, but now, still there are many problems to need to study, such as
Efficiently quick degrading bacteria enables it to be commercialized if preservation of bacteria strain for what selection and breeding collocation, how to remove kitchen garbage degradation
In the process for the pollution of environment, multiple requirements etc. for kitchen waste compost treatment scale in society how are adapted to.
Invention content
It for overcome the deficiencies in the prior art, can the purpose of the present invention is to provide a kind of Geobacillus stearothermophilus
To the higher starch of content in kitchen garbage and albumen fast degradation and the generation of stink can be inhibited, it can be used for kitchen
Quick bio minimizing and the stabilisation of kitchen garbage are realized in the quick processing of rubbish.
To solve the above problems, the technical solution adopted in the present invention is as follows：
A kind of Geobacillus stearothermophilus (Bacillus stearothermophilus) A21, deposit number are
CCTCC NO：M2018137,16S rDNA sequences such as SEQ ID NO：Shown in 1.
As further embodiment, the bacterial strain of Geobacillus stearothermophilus of the present invention is on starch culture-medium
The single bacterium colony of baby pink, bacterium colony surface is smooth, glossy, culture to the slightly raised or sunken irregular pleat in later stage surface
Wrinkle, bacterium colony are sticky.Thalline is rod-short, can produce gemma, and gemma is rice-shaped.
Second object of the present invention is to provide a kind of Geobacillus stearothermophilus liquid bacterial agent, which contains this
The invention Geobacillus stearothermophilus, concrete scheme are as follows：
A kind of Geobacillus stearothermophilus liquid bacterial agent contains Geobacillus stearothermophilus in the microbial inoculum
A concentration of the 10 of (Bacillus stearothermophilus) A219CFU/mL-1010CFU/mL。
Third object of the present invention is to provide a kind of preparation method of Geobacillus stearothermophilus liquid bacterial agent, wrap
It includes
Fermentation step：Bacillus amyloliquefaciens (the Bacillus that will be frozen with tablet activation medium
Stearothermophilus) A21 strains carry out plate streaking, 35-40 DEG C of overnight incubation；
The preparation process of fermentation liquid seed：The good bacterium colony of picking plated growth is in seed culture medium, 35-40 DEG C,
180r/min overnight incubations, obtain liquid seeds；
The step of preparing liquid bacterial agent：Aforesaid liquid seed fermentation medium inoculum concentration 5-10%, is placed in 30-37 DEG C,
150-220r/min, ferment 12-24h, obtains Geobacillus stearothermophilus liquid bacterial agent.
As further embodiment, tablet activation medium of the present invention includes beef extract 3g/L, peptone 10g/
L, NaCl 15g/L, agar 20g/L；The pH of tablet activation medium is 7.2-7.4.
As further embodiment, seed culture medium of the present invention include beef extract 3g/L, peptone 10g/L,
NaCl 15g/L, agar 20g/L；The pH of seed culture medium is 7.2-7.4.
As further embodiment, fermentation medium of the present invention includes beef extract 10g/L, soluble starch 10g/
L, yeast extract 5g/L, KH2PO4 1g/L、MgSO4·7H2O 0.2g/L；Fermentation medium pH 7.0-7.2.
Fourth object of the present invention is to provide a kind of Geobacillus stearothermophilus solid fungicide, by the solid fungicide
It is mixed by Geobacillus stearothermophilus liquid bacterial agent of the present invention and the decomposed dry product of rubbish from cooking, wherein thermophilic
The mass ratio of fatty soil bacillus liquid microbial inoculum and the decomposed dry product of rubbish from cooking is (8-12): 35.
Since Geobacillus stearothermophilus of the present invention has the activity of degradation kitchen garbage, the present invention is also
Provide application of the Geobacillus stearothermophilus in handling kitchen garbage.Preferably concrete application method is：It will be pending
Kitchen garbage pick bone, polybag after suitably crush, rubbish from cooking mixture is made in centrifugal dehydration, mixed in rubbish from cooking
It closes and the solid fungicide containing Geobacillus stearothermophilus is added in material, addition is the 8-15% of garbage weight, is stirred
It ferments respectively at 20-60 DEG C after uniformly, primary ventilation stirring was carried out per 4-5 hours.
Compared with the existing technology, beneficial effects of the present invention are as follows：
1. the Geobacillus stearothermophilus activity that the present invention is screened is high, highly active protein hydrolase and amylolytic enzyme
Activity, and acid can be produced during degradable starch, there is tolerance to high temperature；
2. Geobacillus stearothermophilus of the present invention is to the quick place that can be used for kitchen garbage of kitchen garbage
Reason, realizes quick bio minimizing and the stabilisation of kitchen garbage, while solving kitchen garbage stink in biological treatment process
The problem of release；Weight-loss ratio is higher, Geobacillus stearothermophilus make kitchen garbage be reduced ability with the raising of temperature and by
Cumulative strong, when temperature is increased to 60 DEG C, the decrement ability of Geobacillus stearothermophilus is still up to 80%.
The culture presevation date of the present invention is on March 16th, 2018, and deposit number is CCTCC NO：M2018137.Classification
It is named as：Geobacillus stearothermophilus (Bacillus stearothermophilus) A21.Depositary institution is Chinese Typical Representative
Culture collection, address are Wuhan University of Wuhan, China city, postcode 430072.
Present invention will be further explained below with reference to the attached drawings and examples.
Description of the drawings
Fig. 1 is the primary dcreening operation figure that kitchen garbage is detached in kitchen garbage dirt of the present invention；
Fig. 2 is that Geobacillus stearothermophilus of the present invention grows upper bacterium colony figure in starch culture-medium；
Fig. 3 is the evolution tree graph of Geobacillus stearothermophilus of the present invention；
Fig. 4 is that Geobacillus stearothermophilus of the present invention handles kitchen garbage decrement figure under different vaccination amount；
Fig. 5 is that Geobacillus stearothermophilus of the present invention handles kitchen garbage decrement figure at different temperatures.
Specific implementation mode
A kind of Geobacillus stearothermophilus (Bacillus stearothermophilus) A21 of the present invention,
Deposit number is CCTCC NO：M2018137,16S rDNA sequences such as SEQ ID NO：Shown in 1.
As further embodiment, the bacterial strain of Geobacillus stearothermophilus of the present invention is on starch culture-medium
The single bacterium colony of baby pink, bacterium colony surface is smooth, glossy, culture to the slightly raised or sunken irregular pleat in later stage surface
Wrinkle, bacterium colony are sticky.Thalline is rod-short, can produce gemma, and gemma is rice-shaped.
Second object of the present invention is to provide a kind of Geobacillus stearothermophilus liquid bacterial agent, which contains this
The invention Geobacillus stearothermophilus, concrete scheme are as follows：
A kind of Geobacillus stearothermophilus liquid bacterial agent contains Geobacillus stearothermophilus in the microbial inoculum
A concentration of the 10 of (Bacillus stearothermophilus) A219CFU/mL-1010CFU/mL。
Third object of the present invention is to provide a kind of preparation method of Geobacillus stearothermophilus liquid bacterial agent, wrap
It includes
Fermentation step：Bacillus amyloliquefaciens (the Bacillus that will be frozen with tablet activation medium
Stearothermophilus) A21 strains carry out plate streaking, 35-40 DEG C of overnight incubation；
The preparation process of fermentation liquid seed：The good bacterium colony of picking plated growth is in seed culture medium, 35-40 DEG C,
180r/min overnight incubations, obtain liquid seeds；
The step of preparing liquid bacterial agent：Aforesaid liquid seed fermentation medium inoculum concentration 5-10%, is placed in 30-37 DEG C,
150-220r/min, ferment 12-24h, obtains Geobacillus stearothermophilus liquid bacterial agent.
As further embodiment, tablet activation medium of the present invention includes beef extract 3g/L, peptone 10g/
L, NaCl 15g/L, agar 20g/L；The pH of tablet activation medium is 7.2-7.4.
As further embodiment, seed culture medium of the present invention include beef extract 3g/L, peptone 10g/L,
NaCl 15g/L, agar 20g/L；The pH of seed culture medium is 7.2-7.4.
As further embodiment, fermentation medium of the present invention includes beef extract 10g/L, soluble starch 10g/
L, yeast extract 5g/L, KH2PO4 1g/L、MgSO4·7H2O 0.2g/L；Fermentation medium pH 7.0-7.2.
Fourth object of the present invention is to provide a kind of Geobacillus stearothermophilus solid fungicide, by the solid fungicide
It is mixed by Geobacillus stearothermophilus liquid bacterial agent of the present invention and the decomposed dry product of rubbish from cooking, wherein thermophilic
The mass ratio of fatty soil bacillus liquid microbial inoculum and the decomposed dry product of rubbish from cooking is (8-12): 35.
Since Geobacillus stearothermophilus of the present invention has the activity of degradation kitchen garbage, the present invention is also
Provide application of the Geobacillus stearothermophilus in handling kitchen garbage.Preferably concrete application method is：It will be pending
Kitchen garbage pick bone, polybag after suitably crush, rubbish from cooking mixture is made in centrifugal dehydration, mixed in rubbish from cooking
It closes and the solid fungicide containing Geobacillus stearothermophilus is added in material, addition is the 8-15% of garbage weight, is stirred
It ferments respectively at 20-60 DEG C after uniformly, primary ventilation stirring was carried out per 4-5 hours.
It is specific embodiment of the present invention below, in the following embodiments, used culture medium is listed below respectively：
Protein free medium：Contain skimmed milk power 50g/L, soluble starch 10g/L, yeast extract 5g/L, KH2PO41g/L,
MgSO4·7H2O 0.2g/L, agar 20g/L, pH 7.0-7.2.
Starch culture-medium：Contain soluble starch 20g/L, peptone 5g/L, beef extract 5g/L, NaCl 5g/L, agar
20g/L, pH 7.0-7.2.
Tablet/seed culture medium：Contain meat extract 3g/L, peptone 10g/L, NaCl 15g/L, agar 20g/L, pH 7.2-
7.4。
Fermentation medium：Contain beef extract 10g/L, soluble starch 10g/L, yeast extract 5g/L, KH2PO41g/L,
MgSO4·7H2O 0.2g/L, pH 7.0-7.2.
Embodiment 1：The separation screening of Geobacillus stearothermophilus
The separation process of Geobacillus stearothermophilus of the present invention：Test specimen is acquired from Nanjing University's dining room kitchen garbage
In the splendid attire bucket of carrier loader.Remaining kitchen garbage dirt is scraped out of bucket, loaded in plastic sealing bag, storage is at room temperature.Cause
Sample is relatively insoluble in water, can not detach in water completely so that degradation bacteria negligible amounts in obtained dilution, therefore this use is straight
Connect the mode of solid separation.The sample for taking diameter to be about 1cm from the kitchen garbage of acquisition is connected to starch and protein free medium
Center is placed in 37 DEG C of incubator and cultivates, and observes the growing state of bacterium colony.If with mould in culture medium, to bacterium into
Nystatin is added when can pollute, therefore detach when row scribing line separation in culture medium used to press down mycostatic growth.
To the bacterium colony of growth, the larger scribing line of picking morphological differences isolates and purifies, and numbers and preserves after obtaining pure culture
In slant medium.Directly the placement of kitchen garbage dirt is coated on protein degradation and starch culture-medium, after growing bacterium colony,
With oese picking colony, separation of crossing on protein degradation and starch degradation culture medium respectively, to obtain the pure of single bacterium colony
Bacterium.Meanwhile starch degradation and protein degradation transparent circle are observed, picking has protein and starch the list of preferable degradation capability
Culture and domestication mutagenesis is further purified to improve its degradation property in bacterium colony.
Purifying culture：The single bacterium colony of acquisition is crossed on tablet activation medium, after 37 DEG C of cultures for 24 hours, picking single bacterium
It falls.Each single bacterium colony is individually crossed on one flat plate and is cultivated, checks the unicity of bacterium colony, until it is identical to obtain colonial morphology
Single bacterium colony, that is, complete purifying culture.
To the single bacterium that scribing line obtains after purification, starch degradation culture medium is seeded in using the method that point connects and protein degradation is trained
It supports on base, carries out the measurement of strains for degrading ability, after being cultivated for 24 hours at 37 DEG C, measure colony diameter (d) and transparent loop diameter
(D) measure and calculation D/d carries out the primary dcreening operation of starch and protein degradation ability, selects while having starch and protein degradation ability
Bacterial strain is in case secondary screening.
Experiment primary dcreening operation is obtained into 20 plants of bacterial strains, carries out the secondary screening of starch and protein degradation ability, share 5 plants is in 12h
Now preferable starch and protein degradation performance (transparent circle formed on starch and albumen tablet is big).Choose simultaneously in starch and
Protein culture medium forms the maximum bacterial strain of transparent circle, is identified as Geobacillus stearothermophilus (Bacillus
Stearothermophilus) A21, deposit number are CCTCC NO：M2018137, Main Biological G+ are forming sediment
It is the single bacterium colony of baby pink on powder culture medium, bacterium colony surface is smooth, glossy, and culture is slightly raised or sunken to later stage surface
Irregular fold, bacterium colony is sticky；Thalline is rod-short, can produce gemma, and gemma is rice-shaped.The 16S rDNA sequences of the bacterial strain
Row such as SEQ ID NO：Shown in 1, the sequence of 16S rDNA is compared, and is obtained such as the evolution in Fig. 3 after doing evolutionary analysis
Tree.
Embodiment 2：The preparation of Geobacillus stearothermophilus liquid bacterial agent
Contain the concentration of Geobacillus stearothermophilus (Bacillus stearothermophilus) A21 in the microbial inoculum
It is 109CFU/mL-1010CFU/mL；Preparation method, including
Fermentation step：Bacillus amyloliquefaciens (the Bacillus that will be frozen with tablet activation medium
Stearothermophilus) A21 strains carry out plate streaking, 35-40 DEG C of overnight incubation；
The preparation process of fermentation liquid seed：The good bacterium colony of picking plated growth is in seed culture medium, 35-40 DEG C,
180r/min overnight incubations, obtain liquid seeds；
The step of preparing liquid bacterial agent：Aforesaid liquid seed fermentation medium inoculum concentration 5-10%, is placed in 30-37 DEG C,
150-220r/min, ferment 12-24h, obtains Geobacillus stearothermophilus liquid bacterial agent.
Embodiment 3：The preparation of the solid fungicides of 2-4 containing Geobacillus stearothermophilus
The preparation of the solid fungicides of 2-4 containing Geobacillus stearothermophilus, including
Fermentation step：The strain frozen is subjected to plate streaking, 37 DEG C of overnight incubations with tablet activation medium.
It is prepared by fermentation liquid seed：The good bacterium colony of picking plated growth is in seed culture medium, 37 DEG C, 180r/min cultures
Overnight, liquid seeds are obtained；
Prepare liquid bacterial agent：Inoculum concentration 5-10% is pressed with starch culture-medium, is placed in 30~37 DEG C of constant temperature incubations, 150~
220r/min, ferment 12-24h, obtains Geobacillus stearothermophilus liquid bacterial agent, contains stearothermophilus soil gemma bar in microbial inoculum
Bacterium is 109CFU/mL-1010CFU/mL；
Prepare solid fungicide：The production of the decomposed drying of rubbish from cooking is added into Geobacillus stearothermophilus liquid bacterial agent
Product, addition are the decomposed product 350g Geobacillus stearothermophilus liquid bacterial agent 120g of rubbish from cooking, and stearothermophilus soil is made
Bacillus solid fungicide.
Embodiment 4：Different vaccination amount contains Geobacillus stearothermophilus：Solid fungicide is to the degradation in kitchen garbage
It is suitably crushed after pending kitchen garbage is picked bone, polybag, centrifugal dehydration, rubbish from cooking mixing is made
Material, is added Geobacillus stearothermophilus solid fungicide in rubbish from cooking mixture, and addition is the 1%- of garbage weight
20%, it ferments at 40~45 DEG C after being uniformly mixed, primary ventilation stirring was carried out per 4-5 hours.To processing system after 2 days
System is weighed, and the weight-loss ratio of kitchen garbage is obtained.Weight loss effect is as shown in Figure 4.Within the scope of 1%-20%, stearothermophilus soil bud
The kitchen garbage decrement ability of spore bacillus is gradually increased with the raising of inoculum concentration, when inoculum concentration is 1%-10%, decrement
Rate increases significantly, while the degree of fragmentation of rubbish form increases, when inoculum concentration is 10%-20%, with the increased decrement of inoculum concentration
Rate increase is more slow.
Embodiment 5：Different temperatures solid fungicide containing Geobacillus stearothermophilus is to the degradation in kitchen garbage
It is suitably crushed after pending kitchen garbage is picked bone, polybag, centrifugal dehydration, rubbish from cooking mixing is made
Geobacillus stearothermophilus solid fungicide is added in material in rubbish from cooking mixture, and addition is the 10% of garbage weight, is stirred
It mixes and ferments respectively at 20~60 DEG C after mixing, primary ventilation stirring was carried out per 4-5 hours.To processing system after 2 days
It weighs, obtains the weight-loss ratio of kitchen garbage.Weight loss effect is as shown in Figure 5.Within the scope of 20-50 DEG C, stearothermophilus soil gemma
The kitchen garbage decrement ability of bacillus is gradually increased with the raising of temperature, under the conditions of 20-30 DEG C of medium temperature environment, stearothermophilus
The kitchen garbage decrement ability of native bacillus can be reduced more than 40% in 2 days, under 40-50 DEG C of hot environment, stearothermophilus
Native bacillus can will be reduced more than 80% in 2 days；When temperature is increased to 60 DEG C, the decrement of Geobacillus stearothermophilus
Ability is slightly decreased, but still up to 70%.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Claims (10)
1. a kind of Geobacillus stearothermophilus (Bacillus stearothermophilus) A21, deposit number are
CCTCC NO：M2018137,16S rDNA sequences such as SEQ ID NO：Shown in 1.
2. according to claim 1, which is characterized in that the bacterial strain of the Geobacillus stearothermophilus is in starch culture-medium
The upper single bacterium colony for baby pink, bacterium colony surface is smooth, glossy, cultivates to slightly raised or sunken irregular in later stage surface
Fold, bacterium colony are sticky.Thalline is rod-short, can produce gemma, and gemma is rice-shaped.
3. a kind of Geobacillus stearothermophilus liquid bacterial agent, which is characterized in that contain stearothermophilus soil gemma bar in the microbial inoculum
A concentration of the 10 of bacterium (Bacillus stearothermophilus) A219CFU/mL-1010CFU/mL。
4. a kind of preparation method of Geobacillus stearothermophilus liquid bacterial agent, which is characterized in that including
Fermentation step：Bacillus amyloliquefaciens (the Bacillus that will be frozen with tablet activation medium
Stearothermophilus) A21 strains carry out plate streaking, 35-40 DEG C of overnight incubation；
The preparation process of fermentation liquid seed：The good bacterium colony of picking plated growth is in seed culture medium, 35-40 DEG C, 180r/
Min overnight incubations, obtain liquid seeds；
The step of preparing liquid bacterial agent：Aforesaid liquid seed fermentation medium inoculum concentration 5-10%, is placed in 30-37 DEG C, 150-
220r/min, ferment 12-24h, obtains Geobacillus stearothermophilus liquid bacterial agent.
5. preparation method according to claim 4, which is characterized in that the tablet activation medium includes beef extract 3g/
L, peptone 10g/L, NaCl 15g/L, agar 20g/L；The pH of tablet activation medium is 7.2-7.4.
6. preparation method according to claim 4, which is characterized in that the seed culture medium includes beef extract 3g/L, egg
White peptone 10g/L, NaCl 15g/L, agar 20g/L；The pH of seed culture medium is 7.2-7.4.
7. preparation method according to claim 4, which is characterized in that the fermentation medium include beef extract 10g/L, can
Soluble starch 10g/L, yeast extract 5g/L, KH2PO4 1g/L、MgSO4·7H2O 0.2g/L；Fermentation medium pH 7.0-7.2.
8. a kind of Geobacillus stearothermophilus solid fungicide, which is characterized in that by the stearothermophilus soil bud described in claim 3
Spore bacillus liquid bacterial agent is mixed with the decomposed dry product of rubbish from cooking, wherein Geobacillus stearothermophilus liquid bacterial agent and kitchen
The mass ratio of the remaining decomposed dry product of rubbish is (8-12): 35.
9. application of the Geobacillus stearothermophilus as described in claim 1 in handling kitchen garbage.
10. the application as described in right will require 10, specific method are：Pending kitchen garbage is picked into bone, polybag
Appropriate afterwards to crush, rubbish from cooking mixture is made in centrifugal dehydration, is added in rubbish from cooking mixture and contains stearothermophilus soil bud
The solid fungicide of spore bacillus, addition are the 8-15% of garbage weight, are sent out respectively at 20-60 DEG C after being uniformly mixed
Ferment carried out primary ventilation stirring per 4-5 hours.
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| CN111154690A (en) * | 2020-01-20 | 2020-05-15 | 上海创净生物环保科技有限公司 | Geobacillus thermooleophilic bacillus, microbial inoculum thereof and application of bacillus thermooleophilic bacillus in kitchen waste treatment |
| CN112375704A (en) * | 2020-11-12 | 2021-02-19 | 扬州鑫威环保生物科技有限公司 | Microbial agent for degrading kitchen waste and application method thereof |
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| CN114507625A (en) * | 2022-03-14 | 2022-05-17 | 深圳市微米生物技术有限公司 | Strain combination for producing microbial fertilizer by fermenting kitchen waste and method for producing microbial fertilizer by fermenting kitchen waste |
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