CN109370944A - One plant of bacillus for producing protease and its application in compost - Google Patents
One plant of bacillus for producing protease and its application in compost Download PDFInfo
- Publication number
- CN109370944A CN109370944A CN201811381790.7A CN201811381790A CN109370944A CN 109370944 A CN109370944 A CN 109370944A CN 201811381790 A CN201811381790 A CN 201811381790A CN 109370944 A CN109370944 A CN 109370944A
- Authority
- CN
- China
- Prior art keywords
- bacillus
- strain
- compost
- composting
- temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
- 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
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F7/00—Fertilisers from waste water, sewage sludge, sea slime, ooze or similar masses
-
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Environmental & Geological Engineering (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
- Treatment Of Sludge (AREA)
Abstract
The bacillus for producing protease the invention discloses one plant and its application in compost belong to field of environmental biotechnology.The present invention provides Bacillus hisashii strain B207, which has good sludge protein degradation capability, with other each bacterial strains can good symbiosis, compound proportion is scientific and reasonable.Use the microbial bacterial agent composting compost containing the bacterial strain, heap body reach a high temperature the time required to only 12~for 24 hours, after composting 1.5d, the degradation rate of protein in composting material can be made up to 95.2%-98.9%, it 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 bacillus for producing protease the present invention relates to one plant and its application in compost belong to Environmental Biotechnology neck
Domain.
Background technique
China has urban wastewater treatment firm 3802 in by the end of June, 2015 for cut-off, handles sanitary sewage ability day up to 1.6
Billion cubic meter.With sewage disposal process, the excess sludge generated therewith is about the 0.3%-0.5% of sewage load, China
Generating excess sludge daily is about 40,000,000 tons (moisture content meters by 80%), and is persistently passed with the annual growth of 8%-10%
Increase.Plant nutrient substance rich in sewage treatment excess sludge, such as about 2%-7%, phosphorus 1%- nitrogenous in municipal sludge
5%, potassium 0.1%-0.8%.Potassium content is less in digested sludge, but its content of nitrogen and phosphorous and barnyard manure are similar.Activated sludge
The content of nitrogen and phosphorous is 4-5 times of barnyard manure.In addition, also containing microelements such as sulphur, iron, calcium, sodium, magnesium, zinc, copper, molybdenum, rich in sludge
Rich organic matter and humus.Using sludge composting, existing preferable fertilizer efficiency, and Zinc fractions crumb structure can be made, play improvement
The effect of soil, while realizing the resource utilization of sludge.
However, a large amount of excess sludges of the daily output of sewage treatment plant are deposited in plant area, periodically it is transported to refuse landfill and fills out
It buries or by after the dehydration of excess sludge natural air drying, burns heat supply.On the one hand, these handle disposal options not only higher cost, and
And it is easy that the harmful components in sludge is made to cause secondary pollution to environment in the process.On the other hand, sludge is rich in can
Using organic matter, by taking protein as an example, the protein content of wastewater treatment in China plant excess sludge is 35-60%, including
Microprotein and extracellular polymeric protein two parts.Microorganism accounts for 40% or more of activated sludge dry weight, including bacterium
And microfauna, bacterium accounts for the 90-95% of microorganism, and protein accounts for the 50-60% of bacterial dry mass, protein in microfauna
Content nearly 80%.Exoprotein accounts for total sludge protein about 40%.
For the property and composition of excess sludge, the novel degradation bacteria of screening protein is compounded with other microorganism fungus kinds
Complex micro organism fungicide is obtained, to quickening Composting process, Composting efficiency is improved and improves compost hygienic conditions with important
Meaning.
Summary of the invention
The first purpose of the invention is to provide one plant produce protease Bacillus hisashii strain B207,
China typical culture collection center is preserved on July 13rd, 2018, deposit number is CCTCC No:M 2018473, is protected
Hiding address is China, Wuhan, Wuhan University.The fermentation culture conditions of the bacterial strain are as follows: 30 DEG C -60 DEG C of temperature, ventilatory capacity 0.5-
1vvm, speed of agitator 200-600r/min, dissolved oxygen are greater than 10%, pH value 6.5-7.5.
A second object of the present invention is to provide the microorganisms for containing the Bacillus hisashii strain B207
Microbial inoculum, quantity >=3.0 × 10 of Bacillus hisashii strain B207 in microbial inoculum8CFU/g。
In one embodiment of the invention, Bacillus hisashii strain in the microbial bacterial agent
Quantity >=3.0 × 10 of B2078CFU/mL。
In one embodiment of the invention, the microbial bacterial agent also contains spindle lysine bacillus
(Lysinibacillus fusiformis), library Delhi A Ziweishi Pichia pastoris (Pichia kudriavzevii), vegetables
Bacillus (Bacillus oleronius) and Bacillus thermolactis strain B209；The Bacillus
Thermolactis strain B209, is preserved in China typical culture collection center on July 13rd, 2018, and preservation is compiled
Number be CCTCC No:M 2018474, preservation address be China, Wuhan, Wuhan University.
In one embodiment of the invention, the spindle lysine bacillus (Lysinibacillus
Fusiformis Lysinibacillus fusiformis ATCC 7055) can be selected；The library Delhi A Ziweishi finishes red ferment
Pichia kudriavzevii ATCC 24210, the vegetables bacillus can be selected in female (Pichia kudriavzevii)
Bacillus oleronius DSM 9356 can be selected in (Bacillus oleronius).
In one embodiment of the invention, Bacillus hisashii strain in the microbial bacterial agent
B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus and Bacillus
The ratio between viable count of thermolactis strain B209 is 700-860:3.5-6:1-2.5:700-860:1600-2200,
The total viable count of microbial bacterial agent is 1.0 × 109-1.0×1010CFU/mL or CFU/g.
Third object of the present invention is to provide a kind of method for preparing mentioned microorganism microbial inoculum, the method be by
Bacillus hisashii strain B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetable
Dish bacillus and Bacillus thermolactis strain B209 are seeded in culture medium respectively, obtain Bacillus
Hisashii strain B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus
With the fermentation liquid of Bacillus thermolactis strain B209；The fermentation liquid of acquisition is proportionally mixed, addition hair
The carrier auxiliary material of zymotic fluid quality 0.1~10% obtains liquid microbial microbial inoculum.
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 preparation method of the complex micro organism fungicide is by Bacillus
Hisashii strain B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus
It is mixed in proportion with Bacillus thermolactis strain B209 fermentation liquid, addition crushing straw, charing stalk, bran
One or more of skin after heated-air drying, obtains solid union microbial bacterial agent.
In one embodiment of the invention, Bacillus hisashii strain B207, spindle rely ammonia
Sour bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus and Bacillus thermolactis strain
B209 is seeded to respectively in the nutrient broth medium after high-temperature sterilization, and inoculum concentration is the 3%-10% of fermentation medium volume
(v/v), stationary culture 20-40h；The cultivation temperature of the spindle lysine bacillus is 28-37 DEG C；The Bacillus
Hisashii strain B207, vegetables bacillus and Bacillus thermolactis strain B209 culture temperature
Degree is 30-60 DEG C；When viable count >=1 × 108When CFU/mL, obtains Bacillus hisashii strain B207, spins
The fermentation liquid of capitate lysine bacillus, vegetables bacillus and Bacillus thermolactis strain B209.
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, library Delhi A Ziweishi Pichia pastoris is seeded to the horse after high-temperature sterilization
In bell potato dextrose culture-medium, inoculum concentration is 3%-10% (v/v), the stationary culture 20-30h of fermentation medium volume, culture
Temperature is 20-30 DEG C；When viable count is greater than or equal to 1 × 108When CFU/mL, the library Delhi A Ziweishi Pichia pastoris is obtained
Fermentation liquid.
In one embodiment of the invention, the potato dextrose medium is the horse after 120 DEG C of high-temperature sterilizations
Bell potato dextrose 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.
Fourth object of the present invention is to provide the application of the microbial bacterial agent.
In one embodiment of the invention, the application is to promote food factory effluent processing excess sludge and stalk mixed
Close compost.
In one embodiment of the invention, the application includes the following steps:
(1) complex micro organism fungicide and composting material are mixed according to the ratio of 0.1-0.5:100；
(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
Heap temperature is measured with 6:00 in afternoon, when temperature is higher than 55 DEG C, daily turning is primary, with the extension of composting time, heap body
Temperature is begun to decline, and when temperature is lower than 40 DEG C, stops turning, composting terminates.
The Bacillus hisashii strain B207 is also claimed in food, chemical industry, biology, ring in the present invention
The application in border field.
The utility model has the advantages that
(1) the invention discloses the Bacillus hisashii strain B207 of one plant of degradable protein.The bacterial strain
Protease is produced, enzyme activity has preferable protein degradation ability up to 119.8U/mL after the 26h that ferments.
(2) this compost complex micro organism fungicide provided by the invention, can be used for food factory effluent processing excess sludge with
Stalk mixing composting compost preferably realizes the resource utilization of food factory effluent processing excess sludge and stalk hybrid composting.
(3) this complex micro organism fungicide provided by the invention after composting 1.5d, can make the drop of protein in composting material
Solution rate reaches 95.2%-98.9%, and protein degradation rate is 58.2% 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 12-24h by the 56h of control experiment, shorten composting cycle.
(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 58 DEG C of control experiment to 61-67 DEG C, compost sanitary condition is good.
(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 64-90/g and 95.9%-99.0%%, and
Control experiment is respectively 210/g and 88.9%.
Biomaterial preservation
Bacillus hisashii strain B207 has been preserved in Chinese Typical Representative culture guarantor on July 13rd, 2018
Hiding center, deposit number are CCTCC No:M 2018473, and preservation address is China, Wuhan, Wuhan University.
Bacillus thermolactis strain B209 is preserved in Chinese Typical Representative culture on July 13rd, 2018
Object collection, deposit number are CCTCC No:M 2018474, and preservation address is China, Wuhan, Wuhan University.
Specific embodiment
(1) detection method of protein content: being detected using Kjeldahl's method, i.e., under conditions of having catalyst,
Organic nitrogen is all transformed into inorganic ammonium salt with concentrated sulfuric acid sample digestion, then ammonia is converted by ammonium salt under alkaline condition, with water
Steam distillation comes out and is excessive borate absorption, then with standard titration with hydrochloric acid, so that it may calculate the nitrogen quantity in sample, in turn
Obtain protein content.The degradation rate of protein is R (%):
R=[(m1-m2)/m1/ * 100%
Wherein, m1For the quality (mg) of protein in starting material, m2For the matter of protein in composting for a period of time rear material
It measures (mg).
(2) detection method of excrement colibacillus group number: using the detection method (GB/ of excrement colibacillus group number in fertilizer
T19524.1-2004 it) is detected.
(3) detection method of induced worm egg death rate: using the detection method (GB/ of excrement colibacillus group number in fertilizer
T19524.2-2004 it) is detected.In specific embodiment, spindle lysine bacillus, library Delhi A Ziweishi finish red
Yeast and vegetables bacillus select commercialization bacterial strain: Lysinibacillus fusiformis ATCC 7055, Pichia
Kudriavzevii ATCC 24210, Bacillus oleronius DSM 9356, but it is not only restricted to above-mentioned bacterial strains.
The screening of 1 Bacillus hisashii strain B207 of embodiment
(1) ratio of the excess sludge of food factory effluent processing and corn stover 1.5:1 in mass ratio is mixed, inoculation
The Wuxi City, Jiangsu Province area summer rotted leaf of 5% (w/w), the moisture content of mixed material are that 55%, C/N ratio is 25, are uniformly mixed
Composting compost afterwards, every morning 8:00 and 6:00 in afternoon measure 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, sampling；
(2) using the compost sample of step (1) preparation as the sample of bacterial strain screening, compost sample 10g is weighed, is put into and is equipped with
In the conical flask of 90mL aqua sterilisa, 120r/min shaking table shakes 1h, obtains 10-1Dilution, and successively it is made 10-4、10-5With
10-6Three kinds of compost sample dilutions, draw respectively above-mentioned three kinds of dilutions, 100 μ L be spread evenly across nutrient agar,
On Gause I agar medium and potato agar culture medium plate, be respectively placed in 45 DEG C of incubator be inverted culture 1~
7d, then scribing line separation repeatedly, until obtaining pure bacterial strain；
(3) each pure bacterial strain of gelatin differential medium culture is used, if gelatin liquefaction, expression can produce protease, and can be according to bright
Dispergation degree judges the production protease ability of bacterial strain, and it is stronger that Degree of Liquefaction produces protease ability more again, and laying eggs for acquisition is white
The strongest bacterial strain of matter enzyme ability carries out bacterial strain identification.
The identification of 2 Bacillus hisashii strain B207 of embodiment
On nutrient agar plate after 55 DEG C of overnight incubations, ecru, sharp-edged flat and circle are formed
The bacterium colony of shape is in Gram-positive with optical microscopy observation, rod-shaped in single or pairs of cell arrangement, produces gemma, can
Acid is produced using carbohydrate.
It is sequenced using 16SrDNA and carries out taxology Molecular Identification, sequencing result (1493bp) is subjected to Blast in NCBI
It compares, it is found that its 16SrDNA base sequence is similar to hisashii bacillus (Bacillus hisashii strain)
Property highest, be 99%.
In summary morphological feature, physiological and biochemical property and heredity feature, it is possible to identify the bacteria produced proteinase strain is
Hisashii bacillus (Bacillus hisashii strain), is named as hisashii bacillus B207, and
China typical culture collection center is preserved on July 13rd, 2018, culture presevation number is CCTCC No.M 2018473.
By in strain inoculated to nutrient agar plate, then 45 DEG C of culture 26h are seeded to nutrient broth medium
In, fermentation liquid is centrifuged by 45 DEG C of culture 26h, measures prolease activity using Forint phenol method, after measured, proteinase activity is
119.8U/mL。
Food factory effluent is handled excess sludge and corn stover to mix in 1.5:1 ratio, the moisture content of mixed material is
55%, C/N ratio are 30, and the fermentation liquid of 0.3% (by mass) Bacillus hisashii strain B207 and material is mixed
After closing uniformly, composting compost, 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, and when temperature is lower than 40 DEG C, stops turning, takes
Sample measurement.The results show that protein content reduces 90.2% in raw material after composting 1.5d.
3 Bacillus hisashii strain B207 of embodiment activation and culture
Bacillus hisashii strain B207 is seeded on nutrient agar plate, 45 DEG C of cultures
26h.The group of nutrient agar 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 45 DEG C, 160r/
Min shaken cultivation 26h.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 0.5-1.0vvm, and dissolved oxygen is greater than 10%, fermented and cultured 20-30h under the conditions of 30 DEG C -60 DEG C.Fermentation tank culture medium
Group becomes beef extract 3.0g, peptone 10.0g, sodium acetate 30g, potassium dihydrogen phosphate 2.0g, sodium chloride 5.0g, pH 7.0.
The preparation of the liquid complex micro organism fungicide of 4 strain of hisashii containing Bacillus B207 of embodiment
Compost complex micro organism fungicide is by Bacillus hisashii strain B207 fermentation liquid and other strain fermentations
Liquid compounds, it is specific the preparation method is as follows:
(1) Bacillus hisashii strain B207, spindle lysine bacillus, vegetables bacillus with
The fermentation liquid of Bacillus thermolactis strain B209 is by Bacillus hisashii strain B207, spins
Capitate lysine bacillus, vegetables bacillus and Bacillus thermolactis strain B209 are seeded to respectively
In nutrient broth medium after high-temperature sterilization, inoculum concentration is the 3%-10% (v/v) of fermentation medium volume, cultivates 20-
30h；The cultivation temperature of the spindle lysine bacillus is 28-37 DEG C；The Bacillus hisashii strain
The cultivation temperature of B207, vegetables bacillus and Bacillus thermolactis strain B209 are 30-60 DEG C；Work as work
Bacterium number is greater than or equal to 1 × 108When CFU/mL, Bacillus hisashii strain B207 is obtained, spindle relies ammonia
The fermentation liquid of sour bacillus, vegetables bacillus and Bacillus thermolactis strain B209；
(2) fermentation liquid of library Delhi A Ziweishi Pichia pastoris is that library Delhi A Ziweishi Pichia pastoris is seeded to high temperature
In potato dextrose agar after sterilizing, inoculum concentration is the 3%-10% (v/v) of fermentation medium volume, culture
20-30h, cultivation temperature are 20-30 DEG C；When viable count is greater than or equal to 1 × 108When CFU/mL, library Delhi Ah hereby is obtained
The fermentation liquid of Wei Shi Pichia pastoris；
(3) by Bacillus hisashii strain B207 of acquisition, spindle lysine bacillus, library Delhi
The fermentation liquid of A Ziweishi Pichia pastoris, vegetables bacillus and Bacillus thermolactis strain B209 presses one
Certainty ratio mixing, making the ratio between viable count of each strain is 700~860:3.5~6:1~2.5:700~860:1600~2200,
One or more of the commercially available carrier auxiliary material lignite, zeolite, mountain flour for adding quality of fermentation broth 0.1~10%, obtain viable bacteria
Sum is 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 strain of hisashii containing Bacillus B207 of embodiment
By Bacillus hisashii strain B207 of acquisition, spindle lysine bacillus, library Delhi Ah hereby
Wei Shi Pichia pastoris, vegetables bacillus and Bacillus thermolactis strain B209 fermentation liquid according to certain
Ratio mixing, add commercially available crushing straw, charing stalk, one or more of wheat bran, after heated-air drying, included
The solid union microbial bacterial agent of Bacillus hisashii strain B207.
The application of 6 compost complex micro organism fungicide of embodiment
Food factory effluent is handled excess sludge and corn stover to mix in 1.5:1 ratio, the moisture content of mixed material is
55%, C/N ratio are 30, will contain Bacillus hisashii strain B207, spindle lysine bacillus, library Delhi
The ratio between A Ziweishi Pichia pastoris, vegetables bacillus and viable count of Bacillus thermolactis strain B209
The ratio that liquid complex micro organism fungicide and mixed material for 800:4:1.8:780:2000 are 0.3:100 according to quality proportioning
Mixing, microbial inoculum additive amount are 0.3%, and after mixing, composting compost, every morning 8:00 and 6:00 in afternoon measure two heap bodies
Temperature, when temperature be higher than 55 DEG C when, daily turning is primary, and with the extension of composting time, heap temperature is begun to decline, and works as temperature
When degree is lower than 40 DEG C, stop turning, sampling measures compost items quality index.The result shows that heap body reaches needed for maximum temperature
Time is 15h, and maximum temperature is 66 DEG C；After banking up 1.5d, protein degradation rate 95.9%；At the end of compost, mixed material
Carbon-nitrogen ratio be 17.6, excrement colibacillus group number and induced worm egg death rate are respectively 77/g and 98.2%.
The application of 7 compost complex micro organism fungicide of embodiment
Food factory effluent is handled excess sludge and corn stover to mix in 2.5:1 ratio, the moisture content of mixed material is
55%, C/N ratio are 25, by Bacillus hisashii strain B207, spindle lysine bacillus, library Delhi Ah
Hereby the ratio between Wei Shi Pichia pastoris, vegetables bacillus and the viable count of Bacillus thermolactis strain B209 are
The liquid complex micro organism fungicide and mixed material of 700:3.5:1:700:1600 is mixed according to the ratio that quality proportioning is 0.5:100
It closes, microbial inoculum additive amount is 0.5%, and 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 heap temperature increases rapidly, reach
For for 24 hours, maximum temperature is 61 DEG C the time required to maximum temperature；After banking up 1.5d, protein degradation rate 95.2%；Compost terminates
When, the carbon-nitrogen ratio of mixed material is 18.1, and excrement colibacillus group number and induced worm egg death rate are respectively 89/g and 95.9%.
The application of 8 compost complex micro organism fungicide of embodiment
Food factory effluent is handled excess sludge and corn stover to mix in 2.5:1 ratio, the moisture content of mixed material is
55%, C/N ratio are 25, by Bacillus hisashii strain B207, spindle lysine bacillus, library Delhi Ah
Hereby the ratio between Wei Shi Pichia pastoris, vegetables bacillus and the viable count of Bacillus thermolactis strain B209 are
The liquid complex micro organism fungicide and mixed material of 780:4:1.8:760:1900 is mixed according to the ratio that quality proportioning is 0.5:100
It closes, microbial inoculum additive amount is 0.5%, and 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 heap temperature increases rapidly, reach
It is 12h the time required to maximum temperature, maximum temperature is 67 DEG C；After banking up 1.5d, protein degradation rate 98.9%；Compost terminates
When, the carbon-nitrogen ratio of mixed material is 12.6, and excrement colibacillus group number and induced worm egg death rate are respectively 64/g and 99.0%.
The application of 9 compost complex micro organism fungicide of embodiment
Food factory effluent is handled excess sludge and corn stover to mix in 2.0:1 ratio, the moisture content of mixed material is
55%, C/N ratio are 27, by Bacillus hisashii strain B207, spindle lysine bacillus, library Delhi Ah
Hereby the ratio between Wei Shi Pichia pastoris, vegetables bacillus and the viable count of Bacillus thermolactis strain B209 are
The solid union microbial bacterial agent and mixed material of 860:6:2.5:860:2200 is mixed according to the ratio that quality proportioning is 0.1:100
It closes, microbial inoculum additive amount is 0.1%, after mixing composting compost, and 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 heap temperature increases rapidly, reach
For for 24 hours, maximum temperature is 61 DEG C the time required to maximum temperature；After banking up 1.5d, protein degradation rate 95.2%；Compost terminates
When, the carbon-nitrogen ratio of mixed material is 18.9, and excrement colibacillus group number and induced worm egg death rate are respectively 90/g and 95.9%.
Comparative example 1
Food factory effluent is handled excess sludge and corn stover to mix in 1.5:1 ratio, the moisture content of mixed material is
55%, C/N ratio be 30, by without Bacillus hisashii strain B207 above-mentioned liquid complex micro organism fungicide with
Mixed material is mixed according to the ratio that quality proportioning is 0.3:100, and microbial inoculum additive amount is 0.3%, wherein spindle lysine bud
Spore bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus and Bacillus thermolactis strain B209
The ratio between viable count be 4:1.8:780:2000, after mixing, composting compost, every morning 8:00 and 6:00 in afternoon measurement two
The temperature of a heap body, when temperature is higher than 55 DEG C, daily turning is primary, with the extension of composting time, under heap temperature starts
Drop stops turning when temperature is lower than 40 DEG C, and sampling measures compost items quality index.The result shows that heap body reaches highest
It is 56h the time required to temperature, maximum temperature is 58 DEG C；After banking up 1.5d, protein degradation rate 58.2%；At the end of compost,
The carbon-nitrogen ratio of mixed material is 21.5, and excrement colibacillus group number and induced worm egg death rate are respectively 210/g and 88.9%.
Comparative example 2
With embodiment 6, difference is specific embodiment, and Bacillus hisashii strain B207, spindle rely
Propylhomoserin bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus and Bacillus thermolactis
The ratio between the viable count of strain B209 is variant, specific as shown in table 1.The result shows that the time required to heap body reaches maximum temperature
45-52h is extended to, maximum temperature is 54-56 DEG C；After banking up 1.5 days, protein degradation rate is only 52.1-62.4%；Compost knot
Shu Shi, the carbon-nitrogen ratio of mixed material are 20.0-23.4, and excrement colibacillus group number and induced worm egg death rate are respectively 210-670/g
With 66.8-79.6%.
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)
1. the Bacillus hisashii strain B207 of one plant of production protease, is preserved in China on July 13rd, 2018
Type Tissue Collection, deposit number are CCTCC No:M 2018473, and preservation address is China, Wuhan, Wuhan University.
2. the microbial bacterial agent containing Bacillus hisashii strain B207 described in claim 1, in microbial inoculum
Quantity >=3.0 × 10 of Bacillus hisashii strain B2078CFU/g or CFU/mL.
3. microbial bacterial agent according to claim 2, which is characterized in that also contain spindle lysine bacillus
(Lysinibacillus fusiformis), library Delhi A Ziweishi Pichia pastoris (Pichia kudriavzevii), vegetables
Bacillus (Bacillus oleronius) and Bacillus thermolactis strain B209.
4. microbial bacterial agent according to claim 3, which is characterized in that Bacillus hisashii strain in microbial inoculum
B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus and Bacillus
The ratio between viable count of thermolactis strain B209 is 700-860:3.5-6:1-2.5:700-860:1600-2200,
The total viable count of microbial bacterial agent is 1.0 × 109-1.0×1010CFU/mL or CFU/g.
5. microbial bacterial agent according to claim 4, which is characterized in that the preparation method comprises the following steps: by Bacillus hisashii
Strain B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus with
Bacillus thermolactis strain B209 is seeded in culture medium respectively, obtains Bacillus hisashii
Strain B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus with
The fermentation liquid of Bacillus thermolactis strain B209；The fermentation liquid of acquisition is mixed in proportion, addition carrier is auxiliary
Material obtains liquid microbial microbial inoculum；The carrier auxiliary material includes but is not limited to one or more of lignite, zeolite, mountain flour.
6. microbial bacterial agent according to claim 4, which is characterized in that the preparation method comprises the following steps: by Bacillus hisashii
Strain B207, spindle lysine bacillus, library Delhi A Ziweishi Pichia pastoris, vegetables bacillus with
Bacillus thermolactis strain B209 fermentation liquid mixes in proportion, addition crushing straw, charing stalk, wheat bran
One or more of, after heated-air drying, obtain solid union microbial bacterial agent.
7. Bacillus hisashii strain B207 described in claim 1 is in food, chemical industry, biology, environmental area
Using.
8. a kind of method for promoting food factory effluent processing excess sludge and stalk hybrid composting, which is characterized in that application contains
Bacillus hisashii strain B207 microbial bacterial agent described in claim 1 carries out compost.
9. according to the method described in claim 8, it is characterized by comprising the following steps:
(1) claim 2~the 6 any microbial bacterial agent and composting material are carried out according to the ratio of 0.1-0.5:100
Mixing；
(2) by mixture composting compost, heap temperature is measured, carries out turning according to temperature change.
10. according to the method described in claim 9, it is characterized in that, the step (2) be by the mixture composting compost,
It respectively measures in every morning and afternoon heap temperature 1~2 time；When temperature is higher than 55 DEG C, daily turning is primary；With the composting time
Extension, heap temperature begins to decline, and when temperature is lower than 40 DEG C, stops turning, composting terminates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811381790.7A CN109370944B (en) | 2018-11-20 | 2018-11-20 | Bacillus capable of producing protease and application of bacillus in compost |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811381790.7A CN109370944B (en) | 2018-11-20 | 2018-11-20 | Bacillus capable of producing protease and application of bacillus in compost |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109370944A true CN109370944A (en) | 2019-02-22 |
| CN109370944B CN109370944B (en) | 2020-09-04 |
Family
ID=65389639
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811381790.7A Active CN109370944B (en) | 2018-11-20 | 2018-11-20 | Bacillus capable of producing protease and application of bacillus in compost |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109370944B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862935A (en) * | 2019-08-05 | 2020-03-06 | 浙江佳河绿环境科技有限公司 | High-temperature-resistant bacillus Gongbenniang and high-temperature compost fermentation method |
| CN114921369A (en) * | 2022-05-19 | 2022-08-19 | 中国水产科学研究院黑龙江水产研究所 | Bacterial strain capable of producing protease and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498407A (en) * | 2014-12-24 | 2015-04-08 | 大地绿源环保科技(北京)有限公司 | Bacillus licheniformis UTM107 producing high-temperature-resistant keratinase and application thereof |
| CN105349462A (en) * | 2015-11-26 | 2016-02-24 | 江苏禾喜生物科技有限公司 | Bacillus tequilensis Hexil and application thereof to composting |
| CN105543149A (en) * | 2016-02-04 | 2016-05-04 | 四川师范大学 | Novel bacillus megaterium and application thereof |
| CN108004169A (en) * | 2017-12-18 | 2018-05-08 | 广州王道生物科技有限公司 | Bacillus licheniformis ZL-1 and its application |
-
2018
- 2018-11-20 CN CN201811381790.7A patent/CN109370944B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498407A (en) * | 2014-12-24 | 2015-04-08 | 大地绿源环保科技(北京)有限公司 | Bacillus licheniformis UTM107 producing high-temperature-resistant keratinase and application thereof |
| CN105349462A (en) * | 2015-11-26 | 2016-02-24 | 江苏禾喜生物科技有限公司 | Bacillus tequilensis Hexil and application thereof to composting |
| CN105543149A (en) * | 2016-02-04 | 2016-05-04 | 四川师范大学 | Novel bacillus megaterium and application thereof |
| CN108004169A (en) * | 2017-12-18 | 2018-05-08 | 广州王道生物科技有限公司 | Bacillus licheniformis ZL-1 and its application |
Non-Patent Citations (1)
| Title |
|---|
| 王阶平 等: "芽胞杆菌系统分类研究最新进展", 《福建农业学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862935A (en) * | 2019-08-05 | 2020-03-06 | 浙江佳河绿环境科技有限公司 | High-temperature-resistant bacillus Gongbenniang and high-temperature compost fermentation method |
| CN110862935B (en) * | 2019-08-05 | 2021-04-06 | 浙江佳河绿环境科技有限公司 | High-temperature-resistant bacillus Gongbenniang and high-temperature compost fermentation method |
| CN114921369A (en) * | 2022-05-19 | 2022-08-19 | 中国水产科学研究院黑龙江水产研究所 | Bacterial strain capable of producing protease and application thereof |
| CN114921369B (en) * | 2022-05-19 | 2022-11-04 | 中国水产科学研究院黑龙江水产研究所 | Bacterial strain capable of producing protease and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109370944B (en) | 2020-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103232944B (en) | Microorganism bacterium agent for straw and excrement mixed composting | |
| CN101892182B (en) | Bacillus licheniformis and application thereof in promotion of cellulose degradation | |
| CN102051335B (en) | Method for producing microbial ecological agent by using kitchen garbage | |
| CN103232272B (en) | A kind of compound organic microbial fertilizer and manufacture method thereof | |
| CN109402015B (en) | Bacillus amylovora and application thereof | |
| CN103045511B (en) | Cellulase production bacillus licheniformis with phosphate-solubilizing and potassium-releasing function and application of same | |
| CN101696393A (en) | Compound bacterium agent and method for accelerating fast maturity by inoculating compound bacterium agent to compost | |
| AU2021103470A4 (en) | Bacillus subtilis bs40-4 strain and method for composting organic wastes by using the same | |
| CN107653200B (en) | Microbial agent for promoting aerobic composting of dead pig carcasses and application | |
| CN111363684B (en) | Composite microbial inoculum for efficiently degrading wood fibers and application thereof in composting | |
| CN103896646A (en) | Preparation method of microorganism organic fertilizer | |
| CN102432355A (en) | Banana stem organic fertilizer and preparation method thereof | |
| CN109402013B (en) | High-temperature-resistant bacillus subtilis for producing lipase and application of bacillus subtilis in composting | |
| CN107176891A (en) | It is a kind of to promote the biological agent and its production technology of stalk fast degradation | |
| CN105543149B (en) | One plant of new bacillus megaterium and its application | |
| CN105198504A (en) | Microbial fermentation organic fertilizer and preparation method thereof | |
| CN102010824A (en) | Microbial agent for promoting quick decomposition of agricultural wastes and preparation method | |
| CN110055197B (en) | Paenibacillus amyloliquefaciens BREC-10 and microbial inoculum and application thereof | |
| CN109402014B (en) | Bacillus for producing cellulase and application thereof | |
| CN109370944A (en) | One plant of bacillus for producing protease and its application in compost | |
| CN104560817B (en) | Thermophilic bacillus licheniformis UTM102 for producing phytase and application of thermophilic bacillus licheniformis UTM102 | |
| CN112625948B (en) | Bacillus tequilensis S1 with nitrogen fixation function and application thereof in compost | |
| CN108277187A (en) | Microbial bacterial agent for municipal sludge high-performance bio desiccation | |
| CN102898197A (en) | Technology for producing growth promoting bioorganic fertilizer by using algae mud as additive and product | |
| CN110982711B (en) | Method for open production of trichoderma solid strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |