CN104611258A - Bacillus subtilis and biological deodorant containing bacterial strain of bacillus subtilis - Google Patents
Bacillus subtilis and biological deodorant containing bacterial strain of bacillus subtilis Download PDFInfo
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- CN104611258A CN104611258A CN201410843491.6A CN201410843491A CN104611258A CN 104611258 A CN104611258 A CN 104611258A CN 201410843491 A CN201410843491 A CN 201410843491A CN 104611258 A CN104611258 A CN 104611258A
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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
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS 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
- A61L9/00—Disinfection, sterilisation or deodorisation of air
- A61L9/01—Deodorant compositions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS 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
- A61L2209/00—Aspects relating to disinfection, sterilisation or deodorisation of air
- A61L2209/20—Method-related aspects
- A61L2209/21—Use of chemical compounds for treating air or the like
Abstract
The invention relates to the technical field of function microbiological screening, and particularly provides bacillus subtilis ZNX3 with a deodorizing function. The preservation number of bacillus subtilis ZNX3 is CCTCC NO: M2014649; on the one hand, the bacterial strain can effectively reduce the amount of release of foul gases such as ammonia gas and hydrogen sulfide in manure of livestock and poultry, reduces the generation of the foul gases based on the source, and achieves a remarkable effect as the ammonia gas concentration released by the chicken manure on the fifth day is only 6.7% of a control group; on the other hand, the bacterial strain can quickly degrade ammonia gas and hydrogen sulfide in a henhouse, 10 minutes after spraying, the ammonia gas concentration in the henhouse is reduced by 95.7%, and the hydrogen sulfide concentration is reduced by 94.7%. In addition, the bacillus subtilis ZNX3 can effectively reduce the amount of release of foul gases in household garbage and sewage, the removal rates of ammonia gas, hydrogen sulfide and total hydrocarbon are all higher than 95%, and an unexpected technical effect is achieved.
Description
Technical field
The present invention relates to functional microorganism triage techniques field, be specifically related to a kind of subtilis and comprise the biological deodorant of this bacterial strain.
Background technology
Along with the development of mass-producing, intensive animal husbandry, the pollution of the foul smell produced in Production of Livestock and Poultry to environment constantly aggravates.Stench is as one of environmental hazard, olfactory nerve and the trigeminal nerve of people can be stimulated, respiratory centre is produced and poisons, not only great threat is caused to the health of the mankind, cause topsoil, and seriously affect the health of domestic animal (fowl) and the performance of production performance, therefore stock-farms odor pollution more and more causes the attention of people.For ammonia, if in young pig living environment in air the volume fraction of ammonia reach 0.00005, the rate of body weight gain of young pig can decline 12%; Reach 0.0001 or 0.0005 growth rate and will decline 30%; The keratitis of chicken then can be caused when the volume fraction of ammonia reaches 0.00002 in chicken coop air; When reaching 0.00005, the respiratory rate of chicken will decline, and egg productivity reduces.
Repugnant substance generally distributes in an atmosphere, and some can enter water body with waste water and waste residue, not only makes water generation stench, also can affect hydrobiological existence.Except by the stock-farms planning and design of rational nutritional formula, science and feeding and management, the studied person of development and application of reodorant paid attention to.The reodorant of research and development has both at home and abroad reached more than 20 kinds at present, has plenty of directly to join in Animal fecal pollution and carries out deodorizing, as zeolite, green vitriol, calcium phosphate, potassium permanganate, hydrogen peroxide etc.; Has plenty of the object of adding to and being reached deodorizing in livestock and poultry diet by the digestibility improving diet nutrient material, as yucca shidigera extract, zymin, zeolite, gac, acid supplement, urease inhibitor etc.And the development and application of related microorganism reodorant has become a focus in reodorant research in recent years.
Summary of the invention
The object of this invention is to provide subtilis and application thereof that a strain has deodorization functions.The present invention screens and obtains a bacillus subtilis (Bacillus subtilis) from the chicken manure the feace storing pool of poulty house and surrounding soil thereof, fall ammonia deodorizing effect obvious, effectively can reduce the concentration of the foul gass such as ammonia in colony house and hydrogen sulfide.
One aspect of the present invention provides a bacillus subtilis ZNX3 (Bacillus subtilis ZNX3), be preserved in the China typical culture collection center of Wuhan, China Wuhan University on December 19th, 2014, its deposit number is CCTCC NO:M2014649.
One aspect of the present invention provides a kind of biological deodorant, comprises subtilis ZNX3.
Described biological deodorant also comprises any one or two or more the combination in Bacillus pasteurii, yeast, milk-acid bacteria, acetic bacteria, bacillus amyloliquefaciens, D-lactic acid genus bacillus.
Described biological deodorant also comprises N-(n-butyl) thiophosphoric triamide.
The viable bacteria amount of described biological deodorant is no less than 10
7cfu/mL, wherein the viable bacteria amount of subtilis ZNX3 is not less than 70% of total viable bacteria amount.
Present invention also offers the application of above-mentioned biological deodorant in feces of livestock and poultry, rubbish and sewage disposal.
Beneficial effect of the present invention:
The subtilis ZNX3 that the present invention screens, effectively can reduce the burst size of the foul gas such as ammonia, hydrogen sulfide in feces of livestock and poultry on the one hand, reduce the generation of foul gas from source, the ammonia concentration that chicken manure discharged the 5th day time is only 6.7% of control group, Be very effective; On the other hand, subtilis ZNX3 can also degrade rapidly existing ammonia and hydrogen sulfide in hen house, and after spraying subtilis ZNX310min, in hen house, ammonia concentration reduces 95.7%, and concentration of hydrogen sulfide reduces 94.7%.In addition, subtilis ZNX effectively can also reduce the burst size of foul gas in domestic refuse, sewage, to the clearance of ammonia, hydrogen sulfide and total hydrocarbon all higher than 95%, obtains unexpected technique effect.
Subtilis ZNX3 of the present invention not only can be applied to separately the deodorizing process to feces of livestock and poultry, rubbish and sewage, also can have the microorganism of deodorization functions with other and/or chemical substance is carried out composite, and deodorizing effect is also very remarkable.
Concrete embodiment
The present invention is set forth further below in conjunction with specific embodiment.For concrete grammar used in embodiment or material, those skilled in the art on the basis of the technology of the present invention thinking, can carry out conventional replacement according to existing technology and select, and be not limited only to the concrete record of the embodiment of the present invention.
Separation screening and the qualification of ammonia deodorization functions microorganism fall in embodiment 1
1, substratum:
Nutrient agar;
Selective medium:
| Title | Weight (g) |
| NH 4Cl | 0.125 |
| NaCl | 0.25 |
| KH 2PO 4 | 1.6 |
| K 2HPO 4 | 0.5 |
| MnCl 2·2H 2O | 0.05 |
| MgSO 4·7H 2O | 0.25 |
| MoCl 2 | 0.002 |
| Glucose | 2.50 |
| Dibromothymolsulfonphthalein (1% ethanolic soln) | 1.0 |
| Agar | 20.0 |
| Pure water | 1000 |
2, screening method
(1) sample: be selected from the chicken manure in the feace storing pool of Pingdu poulty house, Qingdao City and surrounding soil thereof;
(2) separation purification method: adopt dilution-plate method;
Chicken manure in chicken house feace storing pool and surrounding soil thereof are respectively sampled 10g, mixes, add the mixing of 100mL stroke-physiological saline solution.Getting 1mL suspension adds in the stroke-physiological saline solution of 9mL, and dilution makes concentration gradient be 10 so repeatedly
-4, 10
-5, 10
-6, 10
-7, 10
-8, from 10
-6, 10
-7, 10
-8respectively get 100 microlitres in extent of dilution to be coated with on nutrient agar, under 30 DEG C of conditions, cultivate 3d, observe colony growth situation, choose the bacterium colony line purifying on flat board, obtain bacterial strain 10 strain altogether, called after ZNX1, ZNX2, ZNX3 successively ... ZNX10 preserves, for subsequent use.
By purifying inoculation in the upper line of selective medium (adding purple reindeer moss (0.2/L) before being down flat plate), after 30 DEG C of cultivation 4d, the appropriate BTB solution of uniform fold on flat board, observation bacterium colony and around dyeing circle size.
Choose the large bacterial strain of colony IFA technique loop diameter as primary dcreening operation bacterial strain, be seeded to nutrient broth medium, 30 DEG C, carry out liquid culture 24h under 160r/min condition after, sterile centrifugation, physiological saline is resuspended, obtains bacteria suspension, and viable count is about 106cfu/mL.
Getting 30g chicken manure loads in large watch-glass, be placed in air chamber, get the suspension liquid 5mL of above-mentioned 10 strain bacterial strains respectively, evenly spray and seal behind chicken manure surface, simultaneously to spray the chicken manure of same volume aqua sterilisa as a control group, use ammonia detector to carry out timing to the release conditions of ammonia in air chamber to detect (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 ammonia is only detected as evaluation index), by the ventilation of gas indoor gas emptying analog culture house after each detection.Ammonia concentration in every day air chamber is in table 1.All 500ppm is designated as when exceeding detection line.
The concentration of ammonia in each air chamber in table 1 test period
As can be seen from the data of table 1, after spraying ZNX3 bacterium liquid, in chicken manure, the burst size of ammonia is well below control group, when the 5th day, in its place air chamber, the concentration of ammonia is only 6.7% of control group, thus the ZNX3 bacterium that explanation the present invention screens effectively can reduce the burst size of ammonia in chicken manure, be conducive to the foul gas reducing feces of livestock and poultry generation.
3, the qualification of ZNX3 bacterial strain
1) colony morphology characteristic: cultivate 18 hours on nutrient agar, be rendered as canescence opaque, there is protuberance centre, slightly fold, and edge is irregular.
2) utilize test kit to extract above-mentioned ZNX3 genomic dna, utilize round pcr amplification 16S rRNA sequence, obtaining length after order-checking is the fragment of 1398bp.By BLAST compare of analysis, it is 99% with the 16SrRNA sequence similarity of the many bacillus subtilis announced, and identifies and confirms that this bacterium is subtilis (Bacillus subtilis), consistent with biochemical identification result.
3) by this bacterium called after subtilis ZNX3 (Bacillus subtilis ZNX3), be preserved in the China typical culture collection center of Wuhan, China Wuhan University on December 19th, 2014, its deposit number is CCTCC NO:M2014649.
The effect of embodiment 2 subtilis ZNX3 in hen house deodorizing
Subtilis ZNX3 is activated, enlarged culturing, through liquid state fermentation, collected by centrifugation bacterium mud, make bacteria suspension, make its bacterium amount reach 10
6cfu/mL.Evenly spray this bacteria suspension in hen house, and make spraying range throughout whole hen house as far as possible.
In the different azimuth of hen house, terrain clearance is about 1.5m place, chooses 7 different sampling points, uses ammonia and sulfurated hydrogen detection instrument to measure respectively to spray front and sprays the concentration of ammonia and hydrogen sulfide in hen house after 10min, and calculating the mean concns of ammonia and hydrogen sulfide, concrete outcome is in table 2.
The concentration that table 2 sprays ammonia and hydrogen sulfide in hen house before and after subtilis ZNX3 compares
| Treatment group | The mean concns (ppm) of ammonia | The mean concns (ppm) of hydrogen sulfide |
| Before spraying | 298.95 | 29.65 |
| After spraying 10min | 12.93 | 1.57 |
As can be seen from the data of table 2, after spraying subtilis ZNX310min, in hen house, the concentration of ammonia and hydrogen sulfide all significantly declines, wherein ammonia concentration reduces 95.7%, concentration of hydrogen sulfide reduces 94.7%, thus further illustrate subtilis ZNX3 of the present invention and effectively to degrade ammonia and hydrogen sulfide, deodorizing effect is remarkable.
In addition, confirm through experiment, subtilis ZNX3 of the present invention not only has significant deodorizing effect to feces of livestock and poultry, effectively can also reduce the burst size of foul gas in domestic refuse, sewage, to the clearance of ammonia, hydrogen sulfide and total hydrocarbon all higher than 95%, obtain unexpected technique effect.
Embodiment 3 comprises the deodorizing effect of biological deodorant to chicken manure of subtilis ZNX3
1, reodorant sample preparation
Subtilis ZNX3 is activated, enlarged culturing, through liquid state fermentation, collected by centrifugation bacterium mud, the quality bacterium mud such as to get and be handled as follows respectively:
(1) sample 1: with physiological saline resuspended subtilis ZNX3 bacterium mud, make bacteria suspension, viable bacteria amount is about 10
7cfu/mL;
(2) sample 2: mixed with yeast and/or milk-acid bacteria by subtilis ZNX3 bacterium mud, make plastc ring, viable bacteria total amount is about 10
7cfu/mL, wherein subtilis ZNX3 proportion is not less than 70%;
(3) sample 3: NBPT (N-(n-butyl) thiophosphoric triamide) aqueous solution of propylene glycol being 5% by subtilis ZNX3 bacterium mud and mass volume ratio mixes, and makes suspension, and wherein the viable bacteria amount of subtilis ZNX3 is about 10
7cfu/mL.
2, Deodorization Experiment
Getting 500g chicken manure loads in large watch-glass, be placed in air chamber, above-mentioned three samples are sprayed according to the usage quantity of 8.5mL/ kilogram of ight soil and seals behind chicken manure surface, simultaneously to spray the chicken manure of same volume aqua sterilisa as a control group, use ammonia detector to carry out timing to the ammonia release conditions in air chamber to detect (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 ammonia is only detected as evaluation index), by the ventilation of gas indoor gas emptying analog culture house after each measurement.In air chamber, ammonia concentration is in table 3.500ppm is designated as when exceeding detection line.
The concentration of ammonia in each air chamber in table 3 test period
As can be seen from the data of table 3, compared with control group, sample 1,2,3 burst sizes that all effectively can reduce ammonia in chicken manure, deodorizing effect is remarkable, wherein with the best results of sample 2 and 3, when the 5th day, in its place air chamber, the concentration of ammonia is only 8% and 6% of control group.Thus illustrate, subtilis ZNX3 of the present invention not only can be applied to separately the deodorizing process to feces of livestock and poultry, also can have the microorganism of deodorization functions with other and/or chemical substance is carried out composite, and deodorizing effect is also very remarkable.
The microorganism with deodorization functions comprises Bacillus pasteurii, yeast, milk-acid bacteria, acetic bacteria, bacillus amyloliquefaciens, D-lactic acid genus bacillus etc.Through verification experimental verification, the biological deodorant obtained after the combination of subtilis ZNX3 and any one or two or more mentioned microorganism is composite, effectively can reduce the burst size of foul gas in feces of livestock and poultry, significantly reduce the concentration of foul gas in rubbish, sewage, Be very effective.
Claims (6)
1. a subtilis, is characterized in that, the deposit number of described subtilis is CCTCC NO:M2014649.
2. a biological deodorant, is characterized in that, described biological deodorant comprises subtilis according to claim 1.
3. biological deodorant as claimed in claim 2, is characterized in that, also comprise any one or two or more the combination in Bacillus pasteurii, yeast, milk-acid bacteria, acetic bacteria, bacillus amyloliquefaciens, D-lactic acid genus bacillus.
4. biological deodorant as claimed in claim 2, is characterized in that, also comprise N-(n-butyl) thiophosphoric triamide.
5. the biological deodorant as described in as arbitrary in claim 2-4, it is characterized in that, in described biological deodorant, total viable bacteria amount is no less than 10
7cfu/mL, the viable bacteria amount of subtilis according to claim 1 is not less than 70% of total viable bacteria amount.
6. the application of biological deodorant according to claim 2 in feces of livestock and poultry, rubbish and sewage disposal.
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Cited By (19)
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