CN104611258B - A kind of bacillus subtilis and the biological deodorant comprising the bacterial strain - Google Patents

A kind of bacillus subtilis and the biological deodorant comprising the bacterial strain Download PDF

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CN104611258B
CN104611258B CN201410843491.6A CN201410843491A CN104611258B CN 104611258 B CN104611258 B CN 104611258B CN 201410843491 A CN201410843491 A CN 201410843491A CN 104611258 B CN104611258 B CN 104611258B
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ammonia
bacillus subtilis
hydrogen sulfide
znx3
concentration
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CN104611258A (en
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郑玉莲
苟万里
甄丽
朱彩彩
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QINGDAO WEILAN TIANCHENG BIOLOGICAL TECHNOLOGY Co Ltd
Qingdao Vland Biotech Group Co Ltd
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QINGDAO WEILAN TIANCHENG BIOLOGICAL TECHNOLOGY Co Ltd
Qingdao Vland Biotech Group Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07Bacillus
    • C12R2001/125Bacillus subtilis ; Hay bacillus; Grass bacillus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L9/00Disinfection, sterilisation or deodorisation of air
    • A61L9/01Deodorant compositions
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B09DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09BDISPOSAL OF SOLID WASTE
    • B09B3/00Destroying solid waste or transforming solid waste or contaminated solids into something useful or harmless
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00Treatment of sludge; Devices therefor
    • C02F11/02Biological treatment
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/347Use of yeasts or fungi
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor
    • C12N1/16Yeasts; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L2209/00Aspects relating to disinfection, sterilisation or deodorisation of air
    • A61L2209/20Method-related aspects
    • A61L2209/21Use of chemical compounds for treating air or the like

Abstract

The present invention relates to functional microorganism triage techniques field, a kind of bacillus subtilis ZNX3 with deodorization functions is specifically provided, its deposit number is CCTCC NO:M2014649.On the one hand the bacterial strain can effectively reduce the burst size of the foul gas such as ammonia in feces of livestock and poultry, hydrogen sulfide, the generation of foul gas be reduced from source, the ammonia concentration that chicken manure discharged at the 5th day is only the 6.7% of control group, and effect is notable;On the other hand, the bacterial strain can also degrade rapidly existing ammonia and hydrogen sulfide in hen house, spray after 10min, ammonia concentration reduces 95.7% in hen house, and concentration of hydrogen sulfide reduces 94.7%.In addition, bacillus subtilis ZNX can also effectively reduce the burst size of foul gas in house refuse, sewage, 95% is above to the clearance of ammonia, hydrogen sulfide and total hydrocarbon, unexpected technique effect is obtained.

Description

A kind of bacillus subtilis and the biological deodorant comprising the bacterial strain
Technical field
The present invention relates to functional microorganism triage techniques field, and in particular to a kind of bacillus subtilis and includes the bacterial strain Biological deodorant.
Background technology
With scale, the development of intensive animal husbandry, pollution of the foul smell produced in Production of Livestock and Poultry to environment constantly adds It is acute.Stench can stimulate the olfactory nerves and trigeminal neuralgia of people as one of environmental hazard, produce murder by poisoning to respiratory center, not only Health to the mankind causes great threat, causes atmosphere pollution, and severely impact the healthy of domestic animal (fowl) and raw The performance of performance is produced, therefore animal farm odor pollution more and more causes the attention of people.By taking ammonia as an example, if young pig life cycle The volume fraction of ammonia reaches 0.00005 in air in border, and the rate of body weight gain of young pig can decline 12%;Reach 0.0001 or 0.0005 Growth rate will decline 30%;The keratitis of chicken can then be triggered when the volume fraction of ammonia reaches 0.00002 in chicken coop air;Reach The respiratory rate of chicken will decline when 0.00005, and egg production is reduced.
Odorant is typically distributed in an atmosphere, and some can enter water body with waste water and waste residue, water is occurred stench, The existence of aquatile can also be influenceed.Except the animal farm planning and designing and feeding management by rational nutrient formulation, science, The development and application of deodorant is paid attention to by researcher.The deodorant researched and developed both at home and abroad at present is planted up to more than 20, is had It is directly added in Animal fecal pollution carry out deodorization, such as zeolite, green vitriol, calcium phosphate, potassium permanganate, hydrogen peroxide;Have plenty of It is added in livestock and poultry diet by improving the digestibility of diet nutrient material to reach the purpose of deodorization, such as yucca spp is extracted Thing, enzyme preparation, zeolite, activated carbon, acid supplement, urease inhibitor etc..And about microbial deodorant development and application into A focus in being studied for deodorant in recent years.
The content of the invention
It is an object of the invention to provide one plant of bacillus subtilis with deodorization functions and its application.The present invention is from poultry Screening obtains a bacillus subtilis (Bacillus subtilis) in chicken manure and its surrounding soil in the feace storing pool of field, drops Ammonia deodorizing effect substantially, can effectively reduce the concentration of the foul gas such as ammonia and hydrogen sulfide in colony house.
One aspect of the present invention provide a bacillus subtilis ZNX3 (Bacillus subtilis ZNX3), in The China typical culture collection center that on December 19th, 2014 is preserved in Wuhan, China Wuhan University, its deposit number is CCTCC NO:M2014649。
One aspect of the present invention provides a kind of biological deodorant, includes bacillus subtilis ZNX3.
The biological deodorant also comprising Bacillus pasteurii, saccharomycete, lactic acid bacteria, acetic acid bacteria, bacillus amyloliquefaciens, Any one or two or more combination in D-lactic acid bacillus.
The biological deodorant also includes N-(n-butyl) thiophosphoric triamide.
The viable bacteria amount of the biological deodorant is no less than 107Cfu/mL, wherein bacillus subtilis ZNX3 viable bacteria amount are not Less than the 70% of total viable bacteria amount.
Present invention also offers application of the above-mentioned biological deodorant in feces of livestock and poultry, rubbish and sewage disposal.
Beneficial effects of the present invention:
The bacillus subtilis ZNX3 that the present invention is screened, on the one hand can effectively reduce ammonia in feces of livestock and poultry, hydrogen sulfide Deng the burst size of foul gas, the generation of foul gas is reduced from source, the ammonia concentration that chicken manure discharged at the 5th day is only For the 6.7% of control group, effect is notable;On the other hand, bacillus subtilis ZNX3 can also degrade rapidly existing ammonia in hen house Gas and hydrogen sulfide, are sprayed after bacillus subtilis ZNX310min, and ammonia concentration reduces 95.7%, concentration of hydrogen sulfide in hen house Reduce 94.7%.In addition, bacillus subtilis ZNX can also effectively reduce the burst size of foul gas in house refuse, sewage, 95% is above to the clearance of ammonia, hydrogen sulfide and total hydrocarbon, unexpected technique effect is obtained.
Bacillus subtilis ZNX3 of the present invention can not only be applied individually to any the deodorization to feces of livestock and poultry, rubbish and sewage Processing, also can have the microorganism of deodorization functions and/or chemical substance to be compounded, deodorizing effect is also very notable with other.
Example is embodied
The present invention is expanded on further with reference to specific embodiment.For specific method or material used in embodiment Material, those skilled in the art can be carried out conventional replacement according to existing technology and selected on the basis of the technology of the present invention thinking Select, be not limited solely to the specific record of the embodiment of the present invention.
The separation screening of the drop ammonia deodorization functions microorganism of embodiment 1 and identification
1st, culture medium:
Nutrient agar;
Selective medium:
Title Weight (g)
NH4Cl 0.125
NaCl 0.25
KH2PO4 1.6
K2HPO4 0.5
MnCl2·2H2O 0.05
MgSO4·7H2O 0.25
MoCl2 0.002
Glucose 2.50
Bromthymol blue (1% ethanol solution) 1.0
Agar 20.0
Pure water 1000
2nd, screening technique
(1) sample:Chicken manure and its surrounding soil in the feace storing pool of Qingdao City Pingdu chicken farm;
(2) isolation and purification method:Using dilution-plate method;
Chicken manure and its surrounding soil in chicken house feace storing pool is respectively sampled into 10g, is well mixed, 100mL sterile physiologicals are added Salt solution is mixed.1mL suspensions are taken to add in 9mL sterile saline, so dilution makes concentration gradient be 10 repeatedly-4, 10-5, 10-6, 10-7, 10-8, from 10-6, 10-7, 10-8100 microlitres of coatings on nutrient agar are respectively taken in dilution factor, in 30 DEG C of conditions Lower culture 3d, observes colony growth situation, chooses the bacterium colony line purifying on flat board, 10 plants of bacterial strain is obtained altogether, is named as successively ZNX1, ZNX2, ZNX3 ... ZNX10 are preserved, standby.
Purifying inoculation is rule on selective medium (be down flat before plate and add purple reindeer moss (0.2/L)), 30 DEG C Cultivate after 4d, the appropriate BTB solution of uniform fold on flat board, observation bacterium colony and surrounding dyeing circle size.
The big bacterial strain of colony IFA technique loop diameter is chosen as primary dcreening operation bacterial strain, nutrient broth medium is seeded to, 30 DEG C, Carried out under the conditions of 160r/min after Liquid Culture 24h, sterile centrifugation, physiological saline is resuspended, and obtains bacteria suspension, viable count is about 106cfu/mL。
Take 30g chicken manures to be fitted into large surface ware, be placed in air chamber, the suspension 5mL of above-mentioned 10 plants of bacterial strains is taken respectively, uniformly Spray in being sealed behind chicken manure surface, while the chicken manure to spray same volume aqua sterilisa is as a control group, use ammonia detector The release conditions of ammonia in air chamber are timed detection (in this experimentation, hydrogen sulfide in the foul gas of chicken manure release Amount is few, mainly ammonia, therefore the change in concentration of only detection ammonia is used as evaluation index), by air chamber after detection is finished every time Interior gas emptying analog culture house ventilation.Ammonia concentration in daily air chamber is shown in Table 1.500ppm is designated as during beyond detection line.
In the test period of table 1 in each air chamber ammonia concentration
Can be seen that and sprayed after ZNX3 bacterium solutions from the data of table 1, in chicken manure the burst size of ammonia well below control group, Concentration of ammonia is only the 6.7% of control group in air chamber where it at the 5th day, so as to illustrate the ZNX3 bacterium that the present invention is screened The burst size of ammonia in chicken manure can be effectively reduced, the foul gas of feces of livestock and poultry generation is advantageously reduced.
3rd, the identification of ZNX3 bacterial strains
1) colony morphology characteristic:Cultivated 18 hours on nutrient agar, be rendered as that canescence is opaque, centre has Protuberance, slightly fold, edge are irregular.
2) above-mentioned ZNX3 genomic DNAs are extracted using kit, 16S rRNA sequences is expanded using round pcr, after sequencing Obtain the fragment that length is 1398bp.Compared and analyzed by BLAST, its 16SrRNA with many bacillus subtilis of announcement Sequence similarity is 99%, identifies that the confirmation bacterium is bacillus subtilis (Bacillus subtilis), with biochemical identification result Unanimously.
3) bacterium is named as bacillus subtilis ZNX3 (Bacillus subtilis ZNX3), in December, 2014 The China typical culture collection center for being preserved in Wuhan, China Wuhan University on 19th, its deposit number is CCTCC NO: M2014649。
Application effects of the bacillus subtilis ZNX3 of embodiment 2 in hen house deodorization
Bacillus subtilis ZNX3 is activated, expands culture, through liquid state fermentation, bacterium mud is collected by centrifugation, bacterium is made and hangs Liquid, makes its bacterium amount reach 106cfu/mL.The bacteria suspension is uniformly sprayed in into hen house, and makes spraying range throughout whole as far as possible Hen house.
In the different azimuth of hen house, terrain clearance is about at 1.5m, to choose 7 different sampled points, use ammonia and sulphur Change hydrogen detecting instrument determine respectively sprays before and spray the concentration of ammonia and hydrogen sulfide in hen house after 10min, and calculate ammonia and The mean concentration of hydrogen sulfide, concrete outcome is shown in Table 2.
The concentration that table 2 sprays ammonia and hydrogen sulfide in hen house before and after bacillus subtilis ZNX3 compares
Treatment group The mean concentration (ppm) of ammonia The mean concentration (ppm) of hydrogen sulfide
Before spraying 298.95 29.65
Spray after 10min 12.93 1.57
It can be seen that and sprayed after bacillus subtilis ZNX310min from the data of table 2, ammonia and hydrogen sulfide in hen house Concentration significantly declines, and wherein ammonia concentration reduces 95.7%, and concentration of hydrogen sulfide reduces 94.7%, so that furtherly Bright bacillus subtilis ZNX3 of the present invention can effectively degrade ammonia and hydrogen sulfide, and deodorizing effect is notable.
In addition, being verified by experiments, bacillus subtilis ZNX3 of the present invention not only has to feces of livestock and poultry significantly to be removed Smelly effect, moreover it is possible to effectively reduce the burst size of foul gas in house refuse, sewage, the removal to ammonia, hydrogen sulfide and total hydrocarbon Rate is above 95%, obtains unexpected technique effect.
Deodorizing effect of biological deodorant of the embodiment 3 comprising bacillus subtilis ZNX3 to chicken manure
1st, deodorant sample preparation
Bacillus subtilis ZNX3 is activated, expands culture, through liquid state fermentation, bacterium mud is collected by centrifugation, the quality such as takes Bacterium mud is handled as follows respectively:
(1) sample 1:Bacillus subtilis ZNX3 bacterium muds are resuspended with physiological saline, bacteria suspension are made, viable bacteria amount is about 107cfu/mL;
(2) sample 2:Bacillus subtilis ZNX3 bacterium muds are mixed with saccharomycete and/or lactic acid bacteria, Mixed Microbes are made Suspension, viable bacteria total amount is about 107Cfu/mL, wherein bacillus subtilis ZNX3 proportions are not less than 70%;
(3) sample 3:By the NBPT (N-(n-butyl) thiophosphorics that bacillus subtilis ZNX3 bacterium muds and mass volume ratio are 5% Triamine) aqueous solution of propylene glycol mixing, suspension is made, wherein bacillus subtilis ZNX3 viable bacteria amount is about 107cfu/mL。
2nd, Deodorization Experiment
Take 500g chicken manures to be fitted into large surface ware, be placed in air chamber, by above three sample according to 8.5mL/ kilograms of excrement Usage amount spray in behind chicken manure surface seal, while the chicken manure to spray same volume aqua sterilisa is as a control group, use ammonia Gas detector is timed detection to the ammonia release conditions in air chamber (in this experimentation, in the foul gas of chicken manure release The amount of hydrogen sulfide is few, mainly ammonia, therefore the change in concentration of only detection ammonia is used as evaluation index), measurement every time is finished Gas indoor gas emptying analog culture is given up afterwards and taken a breath.Ammonia concentration is shown in Table 3 in air chamber.500ppm is designated as during beyond detection line.
In the test period of table 3 in each air chamber ammonia concentration
It is can be seen that from the data of table 3 compared with control group, sample 1,2,3 can effectively reduce releasing for ammonia in chicken manure High-volume, deodorizing effect is notable, wherein with the best results of sample 2 and 3, concentration of ammonia is only in air chamber where it at the 5th day The 8% of control group and 6%.So as to illustrate, bacillus subtilis ZNX3 of the present invention can be not only applied individually to any to animal dung Just deodorizing process, also can have the microorganism of deodorization functions and/or chemical substance to be compounded with other, and deodorizing effect is also very Significantly.
Microorganism with deodorization functions includes Bacillus pasteurii, saccharomycete, lactic acid bacteria, acetic acid bacteria, solution starch gemma Bacillus, D-lactic acid bacillus etc..Experiment proves that, bacillus subtilis ZNX3 with any one or two or more it is above-mentioned The biological deodorant obtained after the combination compounding of microorganism, can effectively reduce the burst size of foul gas in feces of livestock and poultry, significantly The concentration of foul gas in rubbish, sewage is reduced, effect is notable.

Claims (6)

1. a kind of bacillus subtilis, it is characterised in that the deposit number of described bacillus subtilis is CCTCC NO: M2014649。
2. a kind of biological deodorant, it is characterised in that described biological deodorant includes the bacillus subtilis described in claim 1 Bacterium.
3. biological deodorant as claimed in claim 2, it is characterised in that also comprising Bacillus pasteurii, saccharomycete, lactic acid Any one or two or more combination in bacterium, acetic acid bacteria, bacillus amyloliquefaciens, D-lactic acid bacillus.
4. biological deodorant as claimed in claim 2, it is characterised in that also comprising N-(n-butyl) thiophosphoric triamide.
5. the biological deodorant as described in claim 2-4 is any, it is characterised in that total viable bacteria amount in described biological deodorant No less than 107Cfu/mL, the viable bacteria amount of the bacillus subtilis described in claim 1 is not less than the 70% of total viable bacteria amount.
6. application of the biological deodorant in feces of livestock and poultry, rubbish and sewage disposal described in claim 2.
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CN107299068A (en) * 2017-05-17 2017-10-27 刘宇 A kind of swine excrement deodorant and preparation method thereof
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