CN109402013A - A kind of high temperature resistant bacillus subtilis of yielding lipase and its application in compost - Google Patents
A kind of high temperature resistant bacillus subtilis of yielding lipase and its application in compost Download PDFInfo
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- CN109402013A CN109402013A CN201811381862.8A CN201811381862A CN109402013A CN 109402013 A CN109402013 A CN 109402013A CN 201811381862 A CN201811381862 A CN 201811381862A CN 109402013 A CN109402013 A CN 109402013A
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- bacillus
- bacillus subtilis
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- 239000002361 compost Substances 0.000 title claims abstract description 48
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 28
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 27
- 102000004882 Lipase Human genes 0.000 title claims abstract description 14
- 108090001060 Lipase Proteins 0.000 title claims abstract description 14
- 239000004367 Lipase Substances 0.000 title claims abstract description 14
- 235000019421 lipase Nutrition 0.000 title claims abstract description 14
- 238000009264 composting Methods 0.000 claims abstract description 34
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 16
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 57
- 241000194108 Bacillus licheniformis Species 0.000 claims description 30
- 239000000463 material Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 26
- 238000000855 fermentation Methods 0.000 claims description 25
- 230000004151 fermentation Effects 0.000 claims description 25
- 241000894120 Trichoderma atroviride Species 0.000 claims description 24
- 238000002156 mixing Methods 0.000 claims description 15
- 241001134775 Lysinibacillus fusiformis Species 0.000 claims description 11
- 235000013305 food Nutrition 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 10
- 239000010802 sludge Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 241000223259 Trichoderma Species 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000010902 straw Substances 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 4
- 229910021536 Zeolite Inorganic materials 0.000 claims description 3
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 3
- 239000003077 lignite Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 239000010457 zeolite Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 241000193764 Brevibacillus brevis Species 0.000 claims 3
- 239000002131 composite material Substances 0.000 claims 2
- 230000003203 everyday effect Effects 0.000 claims 1
- 238000007602 hot air drying Methods 0.000 claims 1
- 239000010806 kitchen waste Substances 0.000 claims 1
- 239000010865 sewage Substances 0.000 claims 1
- 239000004575 stone Substances 0.000 claims 1
- 241000723920 Bacillus subtilis B2 Species 0.000 abstract description 31
- 230000001580 bacterial effect Effects 0.000 abstract description 27
- 239000010813 municipal solid waste Substances 0.000 abstract description 20
- 239000002253 acid Substances 0.000 abstract description 9
- 238000000354 decomposition reaction Methods 0.000 abstract description 9
- 230000015556 catabolic process Effects 0.000 abstract description 5
- 238000006731 degradation reaction Methods 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 230000007613 environmental effect Effects 0.000 abstract 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 abstract 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 20
- 239000004472 Lysine Substances 0.000 description 20
- 244000005700 microbiome Species 0.000 description 18
- 230000000813 microbial effect Effects 0.000 description 17
- 239000003795 chemical substances by application Substances 0.000 description 16
- 230000000855 fungicidal effect Effects 0.000 description 16
- 239000000417 fungicide Substances 0.000 description 16
- 239000002609 medium Substances 0.000 description 15
- 235000019197 fats Nutrition 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- 238000012545 processing Methods 0.000 description 8
- 240000008042 Zea mays Species 0.000 description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 7
- 235000005822 corn Nutrition 0.000 description 7
- 235000015097 nutrients Nutrition 0.000 description 7
- 239000010907 stover Substances 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 244000061456 Solanum tuberosum Species 0.000 description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 description 6
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 230000000996 additive effect Effects 0.000 description 4
- 235000015278 beef Nutrition 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 239000006916 nutrient agar Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000726221 Gemma Species 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000008121 dextrose Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 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 2
- 229920001817 Agar Polymers 0.000 description 2
- 241001468177 Brevibacillus thermoruber Species 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- 239000005851 Trichoderma atroviride (formerly T. harzianum) strains IMI 206040 and T11 Substances 0.000 description 2
- 241000772340 Trichoderma atroviride IMI 206040 Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000010411 cooking Methods 0.000 description 2
- 235000019784 crude fat Nutrition 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 235000019626 lipase activity Nutrition 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000003519 ventilatory effect Effects 0.000 description 2
- 235000015099 wheat brans Nutrition 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000568397 Lysinibacillus Species 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000010791 domestic waste Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000010812 mixed waste Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000001965 potato dextrose agar Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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- 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
- 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
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- 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
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- 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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Abstract
The invention discloses a kind of high temperature resistant bacillus subtilis of yielding lipase and its applications in compost, belong to field of environmental biotechnology.High temperature resistant bacillus subtilis B 2 11 has ability fatty in good degradation oil-containing kitchen garbage in microbial inoculum prepared by the present invention, it can good symbiosis with other each bacterial strains, compound proportion is scientific and reasonable, use the microbial inoculum composting compost, heap body is short the time required to reaching a high temperature, and fat acid decomposition speed is fast and degradation rate is high, not only can effectively accelerate the speed of compost reaction, the innoxious effect of compost can also be improved, there is extremely wide application prospect.
Description
Technical field
The present invention relates to a kind of high temperature resistant bacillus subtilis of yielding lipase and its applications in compost, and it is raw to belong to environment
Object technical field.
Background technique
China human mortality is numerous, is that rubbish generates big country, according to the municipal refuse statistical data for living the portion's of building publication, every year, I
State's municipal refuse yield has been above 200,000,000 tons；The rubbish close to 0.7 hundred million tons is produced there are also more than 1500 a county towns；As for village
In terms of the rubbish of town, since villages and small towns quantity is dispersed very much, temporarily without accurate statistics data.All in all, China's Output of Domestic Waste exists
400000000 tons or more.House refuse most important composition part in China's is rubbish from cooking, and more than 60%, some areas are even up to
70% to 80%, and the main house refuse of American-European countries is paper, rubbish from cooking only accounts for 25%.In addition, kitchen garbage contains
Water rate is high；It is perishable rotten, pollute environment.The content of organic matter of kitchen garbage is high, and aerobic compost processing is to realize its recycling
With innoxious one of important channel.
However, crude fat content is sometimes up to 25% in kitchen garbage, crude fat content is higher, and material viscosity is larger, ventilation
Property and shatter value are deteriorated, and anaerobic environment is also easy to produce in aerobic composting process, is unfavorable for the progress of aerobic composting process.Another party
Face, fat is substantially not soluble in water, and microbial degradation effect is poor, and when temperature is higher, the fat in kitchen garbage is easily emulsified, and can change
It is apt to its contact condition with microorganism, improves degradation efficiency.Therefore, fat acid decomposition bacterium resistant to high temperature, research and development are screened containing difference
The complex micro organism fungicide of microorganism fungus kind and compound proportion is to Composting process, the raising Composting for accelerating oil-containing kitchen garbage
Efficiency and quality of finished are significant.
Summary of the invention
The first purpose of the invention is to provide a kind of high temperature resistant bacillus subtilis (Bacillus of yielding lipase
Subtilis strain) B211, it is preserved in China typical culture collection center on July 13rd, 2018, deposit number is
CCTCC No:M2018475, preservation address are China, Wuhan, Wuhan University.The fermentation culture conditions of the bacterial strain are as follows: temperature 25-
37 DEG C, ventilatory capacity 0.8-1.5vvm, speed of agitator 200-600r/min, dissolved oxygen is greater than 15%, pH value 6.5-7.5.
A second object of the present invention is to provide the microbial bacterial agents for containing above-mentioned high temperature resistant bacillus subtilis B 2 11.
In one embodiment of the invention, the microbial bacterial agent contains bacillus subtilis B 2 11, spindle relies
Propylhomoserin bacillus (Lysinibacillus fusiformis), bacillus licheniformis B105, thermophilic short bud bacillus B205
With Trichoderma atroviride (Trichoderma atroviride).
In one embodiment of the invention, bacillus licheniformis (the Bacillus licheniformis
Strain) B105 is preserved in China typical culture collection center on July 13rd, 2018, and deposit number is CCTCC No:
M 2018470, preservation address are China, Wuhan, Wuhan University.
In one embodiment of the invention, thermophilic short bud bacillus (the Bacillus thermoruber
Strain) B205 is preserved in China typical culture collection center on July 13rd, 2018, and deposit number is CCTCC No:
M 2018472, preservation address are China, Wuhan, Wuhan University.
In one embodiment of the invention, the spindle lysine bacillus (Lysinibacillus
Fusiformis commercialized bacterial strain) can be selected with Trichoderma atroviride (Trichoderma atroviride), including but not limited to
Lysinibacillus fusiformisATCC 7055、Trichoderma atroviride IMI 206040。
In one embodiment of the invention, the bacillus subtilis B 2 11, spindle lysine bacillus,
The ratio between viable count of clothing bacillus B105, thermophilic short bud bacillus B205 and Trichoderma atroviride is 18-28:2-3:0.5-1.5:
800-1200:2.8-3.3。
In one embodiment of the invention, the total viable count of the microbial bacterial agent is 1.0 × 109-1.0×
1010CFU/mL。
In one embodiment of the invention, the total viable count of the microbial bacterial agent is 1.0 × 109-1.0×
1010CFU/g。
Third object of the present invention is to provide the applications of the microbial bacterial agent.
In one embodiment of the invention, the application is for promoting oil-containing kitchen garbage, at food factory effluent
Manage excess sludge and stalk hybrid composting.
In one embodiment of the invention, the microbial bacterial agent the preparation method comprises the following steps: by high temperature resistant withered grass gemma
Bacillus B211, spindle lysine bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and Trichoderma atroviride
It is seeded in culture medium respectively, obtains high temperature resistant bacillus subtilis B 2 11, spindle lysine bacillus, lichens gemma bar
The fermentation liquid of bacterium B105, thermophilic short bud bacillus B205 and Trichoderma atroviride, the fermentation liquid of acquisition is mixed in proportion, and addition carries
Body auxiliary material obtains the liquid complex micro organism fungicide comprising high temperature resistant bacillus subtilis B 2 11.
In one embodiment of the invention, the carrier auxiliary material includes but is not limited to lignite, zeolite, one in mountain flour
Kind is several.
In one embodiment of the invention, the complex micro organism fungicide the preparation method comprises the following steps: by high temperature resistant withered grass
Bacillus B211, spindle lysine bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 with it is dark green
The fermentation liquid of trichoderma mixes in proportion, and one or more of addition crushing straw, charing stalk, wheat bran after heated-air drying, obtain
To the solid union microbial bacterial agent comprising high temperature resistant bacillus subtilis B 2 11.
In one embodiment of the invention, the acquisition high temperature resistant bacillus subtilis B 2 11, spindle lysine
The fermentation liquid of bacillus, bacillus licheniformis B105 and thermophilic short bud bacillus B205 is by high temperature resistant bacillus subtilis
B211, spindle lysine bacillus, bacillus licheniformis B105 and thermophilic short bud bacillus B205 are inoculated with supreme respectively
In nutrient broth medium after temperature sterilizing, inoculum concentration is the 3%-10% (v/v), stationary culture 20- of fermentation medium volume
40h；The high temperature resistant bacillus subtilis B 2 11, spindle lysine bacillus and bacillus licheniformis B105 culture temperature
Degree is 25-37 DEG C；The cultivation temperature of the thermophilic short bud bacillus B205 is 30-60 DEG C；When viable count be greater than or equal to 1 ×
108When CFU/mL, the high temperature resistant bacillus subtilis B 2 11, spindle lysine bacillus, bacillus licheniformis are obtained
The fermentation liquid of B105 and thermophilic short bud bacillus B205.
In one embodiment of the invention, the nutrient broth medium is the nutrient broth culture after high-temperature sterilization
Base.
In one embodiment of the invention, the ingredient of the nutrient broth medium includes beef extract 3.0g, albumen
Peptone 10.0g, sodium chloride 5.0g, water 1000mL.
In one embodiment of the invention, the fermentation liquid for obtaining Trichoderma atroviride is to be inoculated with Trichoderma atroviride respectively
In potato dextrose medium after to high-temperature sterilization, inoculum concentration is the 3%-10% (v/v) of fermentation medium volume, is stood
20-30h is cultivated, cultivation temperature is 20-30 DEG C；When viable count is greater than or equal to 1 × 108When CFU/mL, the dark green wood is obtained
Mould fermentation liquid.
In one embodiment of the invention, the potato dextrose medium is the potato Portugal after high-temperature sterilization
Grape sugar culture-medium.
In one embodiment of the invention, the ingredient of the potato dextrose medium includes potato leaching powder
6.0g, glucose 20.0g, water 1000mL.
In one embodiment of the invention, the application method of the microbial bacterial agent comprises the following steps:
(1) complex micro organism fungicide and composting material are mixed according to the mass ratio of 0.5-1:100；
(2) by mixture composting compost, heap temperature is measured, carries out turning according to temperature change.
In one embodiment of the invention, the step (2) is by the mixture composting compost, every morning 8:
00 and 6:00 in afternoon measures heap temperature, and when temperature is higher than 55 DEG C, daily turning is primary, with the extension of composting time, heap
Temperature is begun to decline, and when temperature is lower than 40 DEG C, stops turning, composting terminates.
Beneficial effects of the present invention:
(1) the invention discloses the high temperature resistant bacillus subtilis B 2s 11 of one plant of yielding lipase.The bacterial strain yielding lipase, and
High temperature resistant has preferable fat acid decomposition ability, has extremely wide market application prospect.
(2) this compost complex micro organism fungicide provided by the invention can be used for oil-containing kitchen garbage, at food factory effluent
Excess sludge and stalk mixing composting compost are managed, preferably realizes the resource utilization of above-mentioned mixed waste.
(3) this complex micro organism fungicide provided by the invention after composting 2d, can make degradation rate fatty in composting material
Up to 81.9%-95.7%, and fat acid decomposition rate is 56.9% in control experiment.
(4) this microbial bacterial agent provided by the invention is with the obvious advantage in composting compost, the heap body that can effectively shorten heating
Time, so that heap body reaches a high temperature, 55 DEG C of required times foreshorten to 16-32h by the 62h of control experiment.
(5) this microbial bacterial agent provided by the invention is with the obvious advantage in composting compost, can effectively improve heap body most
High-temperature, so that the maximum temperature that heap body reaches is improved by 59 DEG C of control experiment to 61-66 DEG C.
(6) this microbial bacterial agent provided by the invention is with the obvious advantage in composting compost, can improve the innoxious of compost
Effect, at the end of compost, excrement colibacillus group number and induced worm egg death rate are respectively 71-93/g and 96.0%-98.7%, and right
According to the facts test respectively 110/g and 91.8%.
Biomaterial preservation
Bacillus subtilis (Bacillus subtilis strain) B211, has been preserved on July 13rd, 2018
State's Type Tissue Collection, deposit number are CCTCC No:M 2018475, and preservation address is Chinese, Wuhan, and Wuhan is big
It learns.
Bacillus licheniformis (Bacillus licheniformis strain) B105, in preservation on July 13 in 2018
In China typical culture collection center, deposit number is CCTCC No:M 2018470, and preservation address is China, and Wuhan is military
Chinese university.
Thermophilic short bud bacillus (Bacillus thermoruber strain) B205 is protected on July 13rd, 2018
It is hidden in China typical culture collection center, deposit number is CCTCC No:M 2018472, and preservation address is Chinese, Wuhan,
Wuhan University.
Specific embodiment
Detection method is referring to China's biological organic fertilizer quality standard (NY 884-2012).
In specific embodiment, spindle lysine bacillus (Lysinibacillus fusiformis) with it is dark green
Commercialized bacterial strain Lysinibacillus fusiformis ATCC can be selected in trichoderma (Trichoderma atroviride)
7055, Trichoderma atroviride IMI 206040, but it is not only restricted to above-mentioned bacterial strains.
The screening of 1 high temperature resistant bacillus subtilis B 2 11 of embodiment
(1) by oil-containing kitchen garbage, the ratio of food factory effluent processing excess sludge and corn stover 0.5:1:1 in mass ratio
Example mixing, is inoculated with the Wuxi City, Jiangsu Province area summer rotted leaf of 5% (w/w), and the moisture content of mixed material is that 55%, C/N ratio is
25, composting compost, every morning 8:00 and 6:00 in afternoon measure heap temperature after mixing, when temperature is higher than 55 DEG C, often
Its turning is primary, and with the extension of composting time, heap temperature is begun to decline, and when temperature is lower than 40 DEG C, stops turning, takes
Sample；
(2) compost sample 10g is weighed, is put into the conical flask equipped with 90mL aqua sterilisa, 120r/min shaking table shakes 1h,
Obtain 10-1Dilution, and successively it is made 10-4、10-5With 10-6Three kinds of compost sample dilutions, draw respectively above-mentioned three kinds it is dilute
100 μ L of liquid is released to be spread evenly across on nutrient agar, Gause I agar medium and potato agar culture medium plate,
It is respectively placed in 55 DEG C of incubator and is inverted 1~7d of culture, then scribing line separation repeatedly, until obtaining pure bacterial strain；
(3) appropriate 1.6% neutral red aqueous solution is directly dripped and is dipped in bacterium colony surface, if being in navy blue, which is to produce
Lipase strain, degradable aliphatic, and the yielding lipase ability of bacterial strain can be judged according to shade, the deeper yielding lipase energy of color
Power is stronger, final to obtain yielding lipase and the strongest pure bacterial strain of fatty ability of degrading.
The identification of 2 high temperature resistant bacillus subtilis B 2 11 of embodiment
On nutrient agar plate after 30 DEG C of overnight incubations, formation ecru, splintery, surface wettability, in
Between protrusion, the irregular bacterium colony in edge, with optical microscopy observation be in Gram-positive, it is rod-shaped, produce gemma.
It is sequenced using 16SrDNA and carries out taxology Molecular Identification, sequencing result (1496bp) is subjected to Blast in NCBI
It compares, finds the similitude of its 16SrDNA base sequence and bacillus subtilis (Bacillus subtilis strain) most
It is high.
In summary morphological feature, physiological and biochemical property and heredity feature, it is possible to identify the yielding lipase bacterial strain is resistance to height
The bacillus subtilis (Bacillus subtilis strain) of temperature, is named as bacillus subtilis B 2 11, and in
It is preserved in China typical culture collection center on July 13rd, 2018, culture presevation number is CCTCC No.M 2018475.
The ability of bacterial strain yielding lipase is verified.By in strain inoculated to nutrient agar plate, 30 DEG C are trained
25h is supported, then scribing line switching is in nutrient broth medium, 30 DEG C of culture 25h.Fermentation liquid is centrifuged, is surveyed using alkali titration
Determine lipase activity, after measured, lipase activity 9.1U/mL.
By oil-containing kitchen garbage, food factory effluent processing excess sludge and the corn stover ratio of 0.75:0.75:1 in mass ratio
Example mixing, the moisture content of mixed material is that 55%, C/N ratio is 30, by 0.3% (by mass) Bacillus subtilis
After mixing, composting compost, every morning 8:00 and 6:00 in afternoon measure two heaps to the fermentation liquid and material of strain B211
The temperature of body, when temperature is higher than 55 DEG C, daily turning is primary, and with the extension of composting time, heap temperature is begun to decline, when
When temperature is lower than 40 DEG C, stop turning, sampling, the results show that fat content reduces 78.1% in raw material after composting 2d.
Above-mentioned steps compost is pressed using other commercialized bacillus subtilises, the results show that when heap temperature is higher than 35
DEG C when, as temperature increases, microbial activity weakens, and after composting 2d, fat content is not substantially reduced in raw material.
3 high temperature resistant bacillus subtilis B 2 of embodiment, 11 actication of culture and culture
High temperature resistant bacillus subtilis B 2 11 is seeded on nutrient agar plate, 30 DEG C of culture 25h.Nutrition fine jade
The group of rouge culture medium becomes beef extract 3.0g, peptone 10.0g, sodium chloride 5.0g.
Activated strain is inoculated in the 250mL shaking flask equipped with 100mL fluid nutrient medium, under the conditions of 30 DEG C, 450r/
Min shaken cultivation 25h.Shake flask medium group becomes beef extract 3.0g, peptone 10.0g, sodium chloride 5.0g, pH 7.0.
By shake flask culture, 3%-10% is seeded in 3L fermentor by volume, speed of agitator 200-600r/min,
Ventilatory capacity is 0.8-1.5vvm, and dissolved oxygen is greater than 15%, fermented and cultured 20-40h under the conditions of 25 DEG C -37 DEG C.Fermentation tank culture medium
Group becomes beef extract 3.0g, peptone 20.0g, glucose 30g, potassium dihydrogen phosphate 2.0g, sodium chloride 5.0g, pH 7.0.
The preparation of the liquid complex micro organism fungicide of 4 bacillus subtilis B 2 containing high temperature resistant of embodiment 11
Compost complex micro organism fungicide by 11 fermentation liquid of high temperature resistant bacillus subtilis B 2 and other bacterial strain fermentation liquors according to
Different viable counts proportion compounds, it is specific the preparation method is as follows:
(1) high temperature resistant bacillus subtilis B 2 11, spindle lysine bacillus, bacillus licheniformis B105 with it is thermophilic
Short bud bacillus B205 is seeded to respectively in the nutrient broth medium after high-temperature sterilization, and inoculum concentration is fermentation medium volume
3%-10% (v/v), cultivate 20-40h；The high temperature resistant bacillus subtilis B 2 11, spindle lysine bacillus with
The cultivation temperature of bacillus licheniformis B105 is 25-37 DEG C；The cultivation temperature of the thermophilic short bud bacillus B205 of heat is 30-
60℃；When viable count is greater than or equal to 1 × 108When CFU/mL, the high temperature resistant bacillus subtilis B 2 11 is obtained, spindle relies
Propylhomoserin bacillus, bacillus licheniformis B105 and thermophilic short bud bacillus B205 fermentation liquid；
(2) fermentation liquid of Trichoderma atroviride is that Trichoderma atroviride is seeded to the potato dextrose agar culture after high-temperature sterilization
In base, inoculum concentration is the 3%-10% (v/v) of fermentation medium volume, cultivates 20-30h, and cultivation temperature is 20-30 DEG C；Work as work
Bacterium number is greater than or equal to 1 × 108When CFU/mL, the fermentation liquid of the Trichoderma atroviride is obtained；
(3) by the high temperature resistant bacillus subtilis B 2 11 of acquisition, spindle lysine bacillus, bacillus licheniformis
The fermentation liquid of B105, thermophilic short bud bacillus B205 and Trichoderma atroviride are mixed in a certain ratio, and make high temperature resistant bacillus subtilis
Bacterium B211, spindle lysine bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and Trichoderma atroviride
The ratio between viable count is 18-28:2-3:0.5-1.5:800-1200:2.8-3.3, adds the commercially available of quality of fermentation broth 0.1~10%
Carrier auxiliary material lignite, zeolite, one or more of mountain flour, obtaining includes high temperature resistant bacillus subtilis B 2 11, spindle
Lysine bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and Trichoderma atroviride total viable count be 1.0
×109-1.0×1010The liquid complex micro organism fungicide of CFU/mL.
The preparation of the solid union microbial bacterial agent of 5 bacillus subtilis B 2 containing high temperature resistant of embodiment 11
By the high temperature resistant bacillus subtilis B 2 11 of acquisition, spindle lysine bacillus, bacillus licheniformis B105,
The fermentation liquid of thermophilic short bud bacillus B205 and Trichoderma atroviride is mixed in a certain ratio, and makes high temperature resistant bacillus subtilis
The work of B211, spindle lysine bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and Trichoderma atroviride
The ratio between bacterium number is 18-28:2-3:0.5-1.5:800-1200:2.8-3.3, addition crushing straw, charing stalk, one in wheat bran
Kind is several, after heated-air drying, obtains the solid union microbial bacterial agent comprising high temperature resistant bacillus subtilis B 2 11.
The application of 6 compost complex micro organism fungicide of embodiment
Oil-containing kitchen garbage, food factory effluent processing excess sludge and corn stover 0.5:1:1 ratio in mass ratio are mixed
It closes, the moisture content of mixed material is that 55%, C/N ratio is 30, will contain high temperature resistant bacillus subtilis B 2 11, spindle lysine bud
The ratio between spore bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and viable count of Trichoderma atroviride are 24:2.4:1:
The liquid complex micro organism fungicide and mixed material of 1045:2.9 is mixed according to the ratio that quality proportioning is 0.3:100, and microbial inoculum adds
Dosage is 0.3%, and after mixing, composting compost, every morning 8:00 and 6:00 in afternoon measure the temperature of two heap bodies, works as temperature
When degree is higher than 55 DEG C, daily turning is primary, and with the extension of composting time, heap temperature is begun to decline, when temperature is lower than 40 DEG C
When, stop turning, sampling measures compost items quality index.The result shows that it is 21h that heap body, which reaches maximum temperature required time,
Maximum temperature is 65 DEG C；After banking up 2d, fat acid decomposition rate is 93.2%；At the end of compost, the carbon-nitrogen ratio of mixed material is 16.9,
Excrement colibacillus group number and induced worm egg death rate are respectively 88/g and 97.0%.
The application of 7 compost complex micro organism fungicide of embodiment
By oil-containing kitchen garbage, food factory effluent processing excess sludge and the corn stover ratio of 0.5:1.1:0.9 in mass ratio
Example mixing, the moisture content of mixed material is that 55%, C/N ratio is 25, will contain high temperature resistant bacillus subtilis B 2 11, spindle relies ammonia
The ratio between sour bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and viable count of Trichoderma atroviride are 18:2:
The liquid complex micro organism fungicide and mixed material of 0.5:800:2.8 is mixed according to the ratio that quality proportioning is 0.5:100, microbial inoculum
Additive amount is 0.5%, and after mixing, composting compost, every morning 8:00 and 6:00 in afternoon measure the temperature of two heap bodies, when
When temperature is higher than 55 DEG C, daily turning is primary, and with the extension of composting time, heap temperature is begun to decline, when temperature is lower than 40
DEG C when, stop turning, sampling, measure compost items quality index.The result shows that heap temperature increases rapidly, reach the highest temperature
It is 26h the time required to degree, maximum temperature is 63 DEG C；After banking up 2d, fat acid decomposition rate is 88.6%；At the end of compost, mixture
The carbon-nitrogen ratio of material is 17.3, and excrement colibacillus group number and induced worm egg death rate are respectively 90/g and 96.5%.
The application of 8 compost complex micro organism fungicide of embodiment
By oil-containing kitchen garbage, food factory effluent processing excess sludge and the corn stover ratio of 0.5:1.1:0.9 in mass ratio
Example mixing, the moisture content of mixed material is that 55%, C/N ratio is 25, will contain high temperature resistant bacillus subtilis B 2 11, spindle relies ammonia
The ratio between sour bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and viable count of Trichoderma atroviride are 24:
The liquid complex micro organism fungicide and mixed material of 2.4:1:1045:2.9 is mixed according to the ratio that quality proportioning is 0.5:100,
Microbial inoculum additive amount is 0.5%, and after mixing, composting compost, every morning 8:00 and 6:00 in afternoon measure the temperature of two heap bodies
Degree, when temperature is higher than 55 DEG C, daily turning is primary, and with the extension of composting time, heap temperature is begun to decline, when temperature is low
When 40 DEG C, stop turning, sampling measures compost items quality index.The result shows that heap temperature increases rapidly, reach most
It is 16h the time required to high-temperature, maximum temperature is 66 DEG C；After banking up 2d, fat acid decomposition rate is 95.7%；At the end of compost, mix
The carbon-nitrogen ratio for closing material is 11.2, and excrement colibacillus group number and induced worm egg death rate are respectively 71/g and 98.7%.
The application of 8 compost complex micro organism fungicide of embodiment
By oil-containing kitchen garbage, food factory effluent processing excess sludge and the corn stover ratio of 0.5:1.1:0.9 in mass ratio
Example mixing, the moisture content of mixed material is that 55%, C/N ratio is 25, will contain high temperature resistant bacillus subtilis B 2 11, spindle relies ammonia
The ratio between sour bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and viable count of Trichoderma atroviride are 28:3:
The solid union microbial bacterial agent and mixed material of 1.5:1200:3.3 is mixed according to the ratio that quality proportioning is 0.1:100, bacterium
Agent additive amount is 0.1%, after mixing composting compost, and every morning 8:00 and 6:00 in afternoon measure the temperature of two heap bodies,
When temperature is higher than 55 DEG C, daily turning is primary, and with the extension of composting time, heap temperature is begun to decline, when temperature is lower than
At 40 DEG C, stop turning, sampling measures compost items quality index.The result shows that heap temperature increases rapidly, reach highest
It is 32h the time required to temperature, maximum temperature is 61 DEG C；After banking up 2d, fat acid decomposition rate is 81.9%；At the end of compost, mixing
The carbon-nitrogen ratio of material is 18.8, and excrement colibacillus group number and induced worm egg death rate are respectively 93/g and 96.0%.
Comparative example 1
Oil-containing kitchen garbage, food factory effluent processing excess sludge and corn stover 0.5:1:1 ratio in mass ratio are mixed
It closes, the moisture content of mixed material is that 55%, C/N ratio is 30, and the above-mentioned liquid without high temperature resistant bacillus subtilis B 2 11 is answered
It closes microbial bacterial agent and mixed material to mix according to the ratio that quality proportioning is 0.3:100, microbial inoculum additive amount is 0.3%, wherein
Spindle lysine bacillus, bacillus licheniformis B105, thermophilic short bud bacillus B205 and Trichoderma atroviride viable count
The ratio between be 2.4:1:1045:2.9, after mixing, composting compost, every morning 8:00 and 6:00 in afternoon measure two heap bodies
Temperature, when temperature is higher than 55 DEG C, daily turning is primary, and with the extension of composting time, heap temperature is begun to decline, and works as temperature
When lower than 40 DEG C, stop turning, sampling measures compost items quality index.
The result shows that it is 62h that heap body, which reaches maximum temperature required time, maximum temperature is 59 DEG C；After banking up 2d, fat drop
Solution rate is 56.9%；At the end of compost, the carbon-nitrogen ratio of mixed material is 20.9, and excrement colibacillus group number and induced worm egg death rate are distinguished
For 110/g and 91.8%.
Comparative example 2
With embodiment 6, difference is specific embodiment, bacillus subtilis B 2 11, spindle lysine bacillus
(Lysinibacillus fusiformis strain), bacillus licheniformis B105, thermophilic short bud bacillus B205 and depth
The ratio between viable count of green trichoderma is variant, specific as shown in table 1.The result shows that heap body extends the time required to reaching maximum temperature
To 57-64h, maximum temperature is 49-58 DEG C；After banking up 2 days, fat acid decomposition rate is 58.2%-77.7%；At the end of compost, mix
The carbon-nitrogen ratio for closing material is 18.9-24.6, excrement colibacillus group number and induced worm egg death rate be respectively 230-890/g, 56.8%-
88.0%.
Table 1
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
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