CN106244486B - The bacillus of one high-efficiency degradation nitrogen pollutant and its composite bacteria preparation - Google Patents
The bacillus of one high-efficiency degradation nitrogen pollutant and its composite bacteria preparation Download PDFInfo
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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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- 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
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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
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
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Abstract
The invention belongs to microorganism and its application field more particularly to the bacillus of a high-efficiency degradation nitrogen pollutant, further relates to more than one and state bacillus composite bacteria preparation as main component；Further relate to more than one preparation methods for stating bacillus composite bacteria preparation as main component.The bacillus BBHS-01 of efficient degradation nitrogen pollutant of the invention, for white tail feather bacillus BBHS-01 (Bacillus baekryungensis), preservation, deposit number have been carried out in China Committee for Culture Collection of Microorganisms's common micro-organisms collection on December 7th, 2012 are as follows: CGMCC NO.6939.The advantages of white tail feather bacillus BBHS-01 of the invention is that have effects that efficient degradation breeding water body nitrogen pollutant, can purify water, significantly improve cultivation water environment.
Description
Technical field
The invention patent belongs to the gemma bar of microorganism and its application field more particularly to a high-efficiency degradation nitrogen pollutant
Bacterium, be white tail feather bacillus BBHS-01, can efficient degradation aquaculture system nitrogen pollutant, can apply to the water of aquaculture
Environment conditioning and nitrate nitrogen contamination can purify water, significantly improve cultivation water environment；The invention further relates to more than one state it is white
Tail feather bacillus BBHS-01 composite bacteria preparation as main component；The invention further relates to more than one to state white tail feather bacillus
The preparation method of BBHS-01 composite bacteria preparation as main component.
Background technique
Since the 1980s, under the limited driving with vast consumer market demand of wild resource scarcity, China's water
Aquaculture is produced to grow rapidly, cultivation intensive degree is higher and higher, and at the same time, breeding water body pollution increasingly sharpens, wherein
It is especially prominent with polluted by nitrogen in intensive culture pollution.
Nitrogen in breeding water body mainly exists with molecular nitrogen, organic nitrogen, inorganic nitrogen form.In inorganic nitrogen, ammonia nitrogen
There is consumingly toxic action to cultivation aquatic animal with nitrite nitrogen state nitrogen, or even aquaculture organism can be caused dead.There is research table
Bright, litopenaeus vannamei is respectively 2.667mg/L and 5.551mg/L to the tolerable concentration of ammonia nitrogen and nitrite nitrogen.Furthermore in water body
Nitrogen content is excessively high, will cause the phytoplankton excess growth in breeding water body, and a large amount of oxygen for consuming water body seriously affect cultivation
The normal life and growth of biology.Therefore the ammonia nitrogen and nitrite nitrogen of the high concentration in aquaculture, which become, influences aquaculture organism growth
An important factor for health, restriction cultivation development.
In order to improve the speed of growth of aquiculture animal, excessive bait throwing in leads to the accumulation of a large amount of residual baits, molten in residual bait
The harmful substances such as the organic nitrogen solved are not easy to be decomposed, and the tolerance range for being accumulated over aquaculture organism of Water can be to feeding
It grows animal and generates direct toxic action.
With the development of microbial technique, is improved using beneficial microbe progress microorganism regulation and purified water, by
To more and more concerns and research.Existing research person proposes that microorganism plays a crucial role in cultivating pool, drops
Solution microorganism has the function of purifying water in aquaculture, improves and rehabilitates cultivation water environment.In recent years, using beneficial to degradation
Microorganism is developed into probiotics or composite bacteria preparation product is directly thrown into water body and uses, to improve and rehabilitate cultivation water ring
The research in border is more and more, existing scholar study the composite bacteria preparations such as discovery bacillus can effectively degrade it is organic in water body
Sludge promotes Tilapia mossambica (Tilapia) growth；Separately there is scholar to add capsula Rhodopseudomonas in experiment aquarium
After (Rhodopseudomonas capsulata) 1d, the nitrite and ammonia-nitrogen content measured in water body reduces respectively
70%-81% and 35%-41%；It in addition, there will be scholar to have studied in Tilapia mossambica (Tilapia) breeding process using bacteria preparation
Effect, research finds that ammonia nitrogen and nitrous nitrogen content in water body are substantially reduced, and breeding water body bottom dissolved oxygen increases, cultivation water
It is obviously improved.
Probiotics preparation due to being applied to cultivation water environment regulation has the shortcomings that specific aim and bad adaptability, from
The beneficial bacterial strain of separation screening and bacteria preparation is made based on this in breeding water body, bottom environment, in aquaculture water environment
There is extremely profound significance in regulation and the application repaired.
Summary of the invention
An object of the present invention be to provide it is a kind of can be applied to aquaculture water environment regulation and nitrate nitrogen contamination
Bacterial strain --- white tail feather bacillus BBHS-01 (Bacillus baekryungensis), with efficient degradation breeding water body
The effect of nitrogen pollutant, can purify water, significantly improve cultivation water environment.
In order to solve the above-mentioned technical problem, the present invention uses following technical scheme, the bud of a high-efficiency degradation nitrogen pollutant
Spore bacillus is white tail feather bacillus BBHS-01 (Bacillus baekryungensis), micro- in China on December 7th, 2012
Biological inoculum preservation administration committee common micro-organisms collection has carried out preservation, deposit number are as follows: CGMCC NO.6939 is protected
Hiding address is Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
A kind of bacillus of above-mentioned efficient degradation nitrogen pollutant --- white tail feather bacillus BBHS-01 (Bacillus
Baekryungensis) the application in degradation aquaculture pollutant.
The bacillus --- the degradability of white tail feather bacillus BBHS-01 (Bacillus baekryungensis)
In terms of mainly including degradation to inorganic nitrogen, the bacterial strain screening is from prawn culturing pond.
It is furtherd investigate through inventor, acclimation and screening is separated from the bed mud of prawn culturing pond obtaining above-mentioned can be used as addition
Object can be with the white tail feather bacillus BBHS-01 (Bacillus baekryungensis) of efficient degradation water body inorganic nitrogen.It is described white
The main feature of tail feather bacillus BBHS-01 (Bacillus baekryungensis) is as follows:
1, morphological feature:
Form be it is rod-shaped, can move, can produce gemma, binary fission reproduction, thallus is individually, two-by-two or the gathering of short chain.
2, cultural characteristic:
In seawater 2216E basal medium or shrimp feed medium, (commercially available prawn feed 20g is ground into fine powder, sterilizes old
Seawater 1000ml impregnates centrifuging and taking supernatant after 48h, adds agar 25g, adjusts 7.8,121 DEG C of pH, and sterilize 20min) in (5~45)
It is grown at DEG C, optimum growth temperature range is (18~28) DEG C, and optimum temperature range is (24~28) DEG C, grows pH range
It is 4~10, growth optimal pH range is 7.5~8.0, and the NaCl concentration range of the most suitable growth is 30 ‰~60 ‰, 28 ± 1 DEG C
Colony diameter for 24 hours is cultivated on 2216E basal medium or shrimp feed medium up to 1.8mm or so, bacterium colony is in bacterium colony
Be creamy white, round, flat, opaque, neat in edge, surface relatively dry it is coarse.Life is in the white tail feather bacillus of (0-6) h
Long adjustment period, (6-22) h are growth logarithmic phase, and the speed of growth is obviously accelerated, and in especially (6-10) h, thalli growth is most fast,
Bacterial strain enters growth stationary phase and decline phase after 22h.
3, physiological and biochemical property:
Bacterial strain Gram-positive, spore staining is positive, grease hydrolysis enzyme positive, Starch Hydrolysis enzyme positive, catalase sun
Property, V-P negative, glucose oxidative fermentation produce gas, gelatin positive, Lactose-positive, maltose positive, mannitol feminine gender,
Mannose is positive, sucrose is negative, arabinose is negative, xylose is negative.
4, bacterial strain is identified
According to bacillus --- the systematic growth of the 16SrDNA of white tail feather bacillus BBHS-01 and related strain building
Tree is final to determine that bacterial strain is the bacterium of white tail feather bacillus (Bacillus baekryungensis).
5, degradation characteristic
Experiment shows the bacillus --- white tail feather bacillus BBHS-01 is to the ammonia nitrogen (NH in water body4- N), it is sub-
Nitrate nitrogen (NO2- N) and nitrate nitrogen (NO3- N) removal rate respectively be up to 63.43%, 93.98%, 70.37%, it is seen that the bacterial strain
There is very big application potential in aquaculture.
It is a further object of the present invention to provide more than one to state bacillus composite bacteria preparation as main component, described multiple
Combined bacteria preparation is by white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis with the bacterial population of 6~8:1:1
Ratio is combined.
The bacillus subtilis and bacillus licheniformis are purchased from Qingdao root biotechnology group company.It is described compound
Bacteria preparation can effectively remove the pollutant in breeding water body, improve breeding environment.Through inventor repeatedly studies have shown that by upper
State each bacterial strain with the advantages of bacterial population ratio of 6~8:1:1 mixing be not only strain growth it is fast, without antagonism, but also it is made
The composite bacteria preparation obtained is to the NH in water body4-N、NO2- N and NO3The degradation rate of-N is high.
The present invention also provides a kind of above-mentioned systems with white tail feather bacillus BBHS-01 composite bacteria preparation as main component
Preparation Method, the preparation method of the composite bacteria preparation the following steps are included:
(1) actication of culture and purifying: by white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis point
On 2216E solid medium after not being inoculated into improvement, cultivated 22 hours in 28 DEG C；2216E solid culture after the improvement
The culture medium prescription of base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL, agar 25.0g and liquid microelement
1ml, the Trace Elements of the liquid microelement are as follows: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O,
The MnSO of 0.5g/L4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4, adjust pH and sterilize for 7.6,121 DEG C, 101KPa
30min；Picking single colonie is inoculated into 2216E solid slope culture medium, cultivates 20 hours in 28 DEG C of purifying, resulting test tube slant
Bacterial strain is Purify bacterial strain, and the 2216E solid slope culture medium ingredient is the same as the 2216E solid medium after improvement；
(2) it makes triangular flask liquid spawn: test tube slant bacterial strain is inoculated in the 2216E fluid nutrient medium after improvement respectively
On, it is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask liquid spawn of each bacterial strain；2216E liquid training after the improvement
The culture medium prescription for supporting base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL and liquid microelement 1ml, described
The trace element suite of liquid microelement is divided into: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O, 0.5g/L's
MnSO4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4；
(4) preparation of mixed bacteria liquid: by the triangular flask liquid spawn of cultured each bacterial strain according to white tail feather bacillus
BBHS-01, bacillus subtilis and bacillus licheniformis are inoculated into 2216E fluid nutrient medium with the bacterial population ratio of 6~8:1:1
In, it is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask mixing liquid strain of three bacterial strains；
(5) preparation of composite bacteria preparation:
A. yeast powder, peptone, sodium chloride, bean cake powder, wheat bran skin and water are mixed with the weight ratio of 2:6:1:1:2:10
Uniformly, culture medium solid is made；
B. zeolite powder, peanut meal and medical stone powder are mixed with the weight ratio of 1:1:1, gained mixture is filled out as auxiliary material
Fill object；
C. ooze is crushed with pulverizer, is sieved with 100 mesh sieve, is added in extractor after sieving, be 50% by mass concentration
Alcohol is added in extractor, and alcohol dosage is 10 times of ooze weight, opens stirring, extractor is placed in water bath with thermostatic control shaking table,
30min is extracted under the conditions of 140rpm, 70~80 DEG C, 100 mesh filter to take filtrate, and filter residue, which returns, to be repeated to extract one in extractor
Secondary, 100 mesh filter to take filtrate, merge gained filtrate twice, obtain ooze leachate；
D. weigh each component of following parts by weight: wood fragments consider to be worth doing 12~15 parts, 10~12 parts of wheat straw, 10~12 parts of mulberry leaf,
9~10 parts of duckweed, 9~10 parts of david poplar bark, 5 parts of fish scale, 4~5 parts of balsam pear, 1 part of reed root, 1 part of distiller's dried grain, 1 part of dry ferment, shellfish
1 part of shell powder, load weighted each component is mixed, and the purified water of equivalent is added into mixture, stirred and evenly mixed, is put into glass appearance
Device is protected from light fermentation 56 hours under the conditions of 28 DEG C~30 DEG C, obtains tunning, 100 mesh net filtrations of gained tunning take
Filter resulting fermentation liquid；
E. culture medium solid, ooze leachate, triangular flask mixing liquid strain is equal according to the weight ratio of 4:1:60
Even mixing is subsequently placed in fermentor in 28 DEG C, 180~200rpm of mixing speed, ventilatory capacity 4V/V.min, cultivates 5d, obtain
To mixed fermentation bacterium solution；
F. auxiliary material filler, fermentation liquid, bentonite, mixed fermentation bacterium solution are mixed according to the weight ratio of 1:1~2:1:8~9
It closes, is placed in 50 DEG C of drying machine dryings, crush, composite bacteria preparation is made.
In the d operating procedure of step (5), by 100 mesh net filtrations of gained tunning, the resulting fermentation liquid of filtering is taken
Effect be to prevent the solid residue in tunning from entering breeding water body, avoid bringing secondary pollution to breeding water body.
Preferably, the partial size of the zeolite powder and medical stone powder is 100 mesh, and the bentonitic partial size is 150 mesh.Due to
The size of specific surface area can have an impact adsorption effect, therefore bentonite of the invention is formd with zeolite powder and medical stone powder
The particle of different specific surface areas is formed a team, the experimental results showed that, bentonite is crushed to different grains from zeolite powder and medical stone powder
Diameter is conducive to improve the synergic sorption between bentonite, zeolite powder and medical stone powder, and then improves composite bacteria preparation to water
Produce the degradation rate of breeding pollution object and nitrogen pollutant.
Preferably, in step (4), bacillus BBHS-01, bacillus subtilis and bacillus licheniformis are with 7:1:1's
Bacterial population ratio is inoculated into 2216E fluid nutrient medium.
Preferably, in the d operating procedure of step (5), the parts by weight of weighed each component are as follows: wood fragments consider 15 parts, wheat to be worth doing
12 parts of straw, 10 parts of mulberry leaf, 10 parts of duckweed, 9 parts of david poplar bark, 5 parts of fish scale, 4 parts of balsam pear, 1 part of reed root, 1 part of distiller's dried grain, dry ferment
1 part, 1 part of conch meal.
Preferably, in the f operating procedure of step (5), auxiliary material filler, fermentation liquid, bentonite mixed fermentation bacterium solution according to
The weight ratio of 1:2:1:8 mixes.
In the present invention, composite bacteria preparation is to the NH in water body4-N、NO2- N and NO3The highest degradation rate of-N is respectively
93.27%, 100.00% and 92.09%, the degradation effect of composite bacteria preparation is significantly better than single strain, it is seen that bacillus
BBHS-01, bacillus subtilis and bacillus licheniformis have synergistic effect to the degradation aspect of nitrogen pollutant, improve
White tail feather bacillus BBHS-01 is to the degradation rate of nitrogen pollutant, in addition, composite bacteria preparation of the invention has storage and user
Just the advantages of, has broad application prospects in aquaculture.
Compared with prior art, the beneficial effects of the present invention are: (1) white tail feather bacillus BBHS-01 of the invention is to water
Ammonia nitrogen (NH in body4- N), cultured water (NO2- N) and nitrate nitrogen (NO3- N) removal rate respectively be up to 63.43%,
93.98%, 70.37%, there is very big application potential in aquaculture；(2) by white tail feather bacillus BBHS-01, withered
The advantages of careless bacillus and bacillus licheniformis are mixed with the bacterial population ratio of 6~8:1:1 be not only strain growth it is fast, without short of money
Anti- effect, and obtained composite bacteria preparation is to the NH in water body4-N、NO2- N and NO3The degradation rate of-N is high, in water body
NH4-N、NO2- N and NO3The highest degradation rate of-N is respectively 93.27%, 100.00% and 92.09%, composite bacteria preparation
Degradation effect is better than single strain, it is seen that white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis are to polluted by nitrogen
There is synergistic effect in terms of the degradation of object；(3) composite bacteria preparation of the invention have the advantages that storage and it is easy to use,
It has broad application prospects in aquaculture.
Detailed description of the invention
Fig. 1 is the growth curve of white tail feather bacillus BBHS-01 of the invention；
Fig. 2 is the phylogenetic tree based on 16SrDNA gene order of white tail feather bacillus BBHS-01 and related strain.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.
The main feature of the white tail feather bacillus of embodiment 1 and its degradation experiment to water pollutant
1. the main feature of the white tail feather bacillus BBHS-01 (Bacillus baekryungensis) is as follows:
(1) morphological feature:
Form be it is rod-shaped, can move, can produce gemma, binary fission reproduction, thallus is individually, two-by-two or the gathering of short chain.
(2) cultural characteristic:
In seawater 2216E basal medium or shrimp feed medium, (commercially available prawn feed 20g is ground into fine powder, sterilizes old
Seawater 1000ml impregnates centrifuging and taking supernatant after 48h, adds agar 25g, adjusts 7.8,121 DEG C of pH, and sterilize 20min) in (5~45)
It is grown at DEG C, optimum growth temperature range is (18~28) DEG C, and the optimum temperature range to prawn feed ingredient degradation is (24
~28) DEG C, growth pH range is 4~10, and the optimal pH range of growth and bait degradation is 7.5~8.0, the most suitable growth
NaCl concentration range is 30 ‰~60 ‰, and (28 ± 1) DEG C are cultivated for 24 hours on 2216E basal medium or shrimp feed medium
Colony diameter up to 1.8mm or so, bacterium colony is creamy white in bacterium colony, round, flat, opaque, neat in edge, surface are more dry
It is dry coarse.It is in growth adjustment period in the white tail feather bacillus of (0-6) h, (6-22) h is growth logarithmic phase, and the speed of growth obviously adds
Fastly, especially in (6-10) h, thalli growth is most fast, and bacterial strain enters growth stationary phase after 22h and decline phase, growth curve are shown in
Attached drawing 1.
(3) physiological and biochemical property:
Bacterial strain Gram-positive, spore staining is positive, grease hydrolysis enzyme positive, Starch Hydrolysis enzyme positive, contacts enzyme positive,
V-P negative, glucose oxidative fermentation produce gas, the gelatin positive, Lactose-positive, the maltose positive, mannitol feminine gender, sweet dew
Sugared positive, sucrose feminine gender, arabinose is negative, xylose is negative.
(4) bacterial strain is identified
Bacterium is finally determined according to the phylogenetic tree of the 16SrDNA of white tail feather bacillus BBHS-01 and related strain building
Strain is that (see Fig. 2, wherein BBHS-01 is of the invention for the bacterium of white tail feather bacillus (Bacillus baekryungensis)
The white tail feather bacillus of bacterial strain).
(5) degradation characteristic
Experiment shows the white tail feather bacillus BBHS-01 to the ammonia nitrogen (NH in water body4- N), cultured water (NO2-N)
With nitrate nitrogen (NO3- N) removal rate respectively be up to 63.43%, 93.98%, 70.37%, it is seen that the bacterial strain is in aquaculture
Have very big application potential.
2. degradation of the white tail feather bacillus BBHS-01 (Bacillus baekryungensis) to water pollutant
Experiment:
(1) prawn feed solid plate growing state detects:
White tail feather bacillus BBSH-01 (deposit number are as follows: CGMCC is separated from the bed mud of prawn culturing pond
NO.6939), in 28 ± 1 DEG C of shrimp feed mediums in 20g/L, (commercially available prawn feed 20g is ground into fine powder to the bacterial strain, sterilizing
Chen Haishui 1000ml impregnates centrifuging and taking supernatant after 48h, adds agar 25g, adjusts 7.8,121 DEG C of pH, and sterilize 20min) on growing way it is good,
The colony diameter for cultivating 24 rapidly is grown up to 2mm or so.
(2) the degradation effect detection of prawn feed leachate:
1) white tail feather bacillus BBHS-01 tests the degradation effect of prawn feed leachate
Experiment degradation solution used: being ground into fine powder for commercially available prawn feed 20g, and sterilize Chen Haishui 1000ml, after impregnating 48h
Centrifuging and taking supernatant adds sodium nitrite 0.058g, adjusts pH 7.8, is distributed into 100mL/ bottles, and in 121 DEG C, sterilize 20min.
The object of the activation culture for 24 hours 8000r/min of white tail feather bacillus BBHS-01 is centrifuged 10min, with sterile saline
Lotion precipitates thallus, and is diluted to containing 1 × 106Then bacterium solution is added to pair of 100mL by the bacterium solution of CFU/mL in 1% ratio
In shrimp bait degradation solution, 28 ± 1 DEG C, shake culture 5d under 160r/min, daily timing sampling measure the ammonia nitrogen in sample liquid
(NH4- N), cultured water (NO2- N) and nitrate nitrogen (NO3- N) content, thalli growth amount uses photoelectric turbidimetry with OD600It indicates,
Bacterium is not added as control treatment, each processing sets 3 repetitions.
2) experimental result
Experimental result is shown in Tables 1 and 2 respectively.The results showed that white tail feather bacillus BBHS-01 degrades to prawn feed
Liquid has apparent degradation effect, inorganic in the bait degradation solution that can significantly degrade during the experiment with the growth of the bacterial strain
Nitrogen component, wherein to NH4-N、NO2-N、NO3The highest degradation rate of-N is respectively 63.43%, 93.98%, 70.37%.
The white tail feather bacillus BBHS-01 of table 1 is in different incubation times to the degradation effect (1~5d) of bait degradation solution
The white tail feather bacillus BBHS-01 of table 2 is in different incubation times to the degradation effect (1~5d) of bait degradation solution
The white tail feather bacillus BBHS-01 of embodiment 2, bacillus subtilis and bacillus licheniformis antagonistic experiment
The antagonistic experiment of three bacterial strains: white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis are taken respectively
It crosses with 2216E solid medium, after being placed in 28 DEG C of cultures for 24 hours, picks them separately single colonie and carried out on 2216E solid medium
Cross is crossed two-by-two, by the plate of scribing line after 28 DEG C of cultures for 24 hours, checks strain growth situation, antagonistic experiment scribing line training
It supports the result shows that antagonism is not present in three bacterial strains.
The preparation method of 3 composite bacteria preparation of embodiment
The preparation method of the composite bacteria preparation of the present embodiment the following steps are included:
(1) actication of culture and purifying: by white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis point
On 2216E solid medium after not being inoculated into improvement, cultivated 22 hours in 28 DEG C；2216E solid culture after the improvement
The culture medium prescription of base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL, agar 25.0g and liquid microelement
1ml, the Trace Elements of the liquid microelement are as follows: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O,
The MnSO of 0.5g/L4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4, adjust pH and sterilize for 7.6,121 DEG C, 101KPa
30min；Picking single colonie is inoculated into 2216E solid slope culture medium, cultivates 20 hours in 28 DEG C of purifying, resulting test tube slant
Bacterial strain is Purify bacterial strain, and the 2216E solid slope culture medium ingredient is the same as the 2216E solid medium after improvement；
(2) it makes triangular flask liquid spawn: test tube slant bacterial strain is inoculated in the 2216E fluid nutrient medium after improvement respectively
On, it is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask liquid spawn of each bacterial strain；2216E liquid training after the improvement
The culture medium prescription for supporting base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL and liquid microelement 1ml, described
The trace element suite of liquid microelement is divided into: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O, 0.5g/L's
MnSO4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4；
(4) preparation of mixed bacteria liquid: by the triangular flask liquid spawn of cultured each bacterial strain according to white tail feather bacillus
BBHS-01, bacillus subtilis and bacillus licheniformis are inoculated into 2216E fluid nutrient medium with the bacterial population ratio of 7:1:1,
It is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask mixing liquid strain of three bacterial strains；
(5) preparation of composite bacteria preparation:
A. yeast powder, peptone, sodium chloride, bean cake powder, wheat bran skin and water are mixed with the weight ratio of 2:6:1:1:2:10
Uniformly, culture medium solid is made；
B. zeolite powder and medical stone powder are crushed respectively, and sieved with 100 mesh sieve, by zeolite powder, peanut meal and medical stone powder with
The weight ratio of 1:1:1 mixes, and gained mixture is as auxiliary material filler；
C. ooze is crushed with pulverizer, is sieved with 100 mesh sieve, is added in extractor after sieving, be 50% by mass concentration
Alcohol is added in extractor, and alcohol dosage is 10 times of ooze weight, opens stirring, extractor is placed in water bath with thermostatic control shaking table,
30min is extracted under the conditions of 140rpm, 70~80 DEG C, 100 mesh filter to take filtrate, and filter residue, which returns, to be repeated to extract one in extractor
Secondary, 100 mesh filter to take filtrate, merge gained filtrate twice, obtain ooze leachate；
D. weigh following each component according to following parts by weight: wood fragments consider 15 parts, 12 parts of wheat straw, 10 parts of mulberry leaf, duckweed 10 to be worth doing
Part, 9 parts of david poplar bark, 5 parts of fish scale, 4 parts of balsam pear, 1 part of reed root, 1 part of distiller's dried grain, 1 part of dry ferment, 1 part of conch meal, will weigh
Each component mix, and into mixture be added equivalent purified water, stir and evenly mix, be put into glass container, 28 DEG C~30 DEG C items
It is protected from light fermentation 56 hours under part, obtains tunning, by 100 mesh net filtrations of gained tunning, takes the resulting fermentation of filtering
Liquid；
E. culture medium solid, ooze leachate, triangular flask mixing liquid strain is equal according to the weight ratio of 4:1:60
Even mixing is subsequently placed in fermentor in 28 DEG C, 180~200rpm of mixing speed, ventilatory capacity 4V/V.min, cultivates 5d, obtain
To mixed fermentation bacterium solution；
F., bentonite is crushed to and crossed 150 meshes, auxiliary material filler, fermentation liquid, bentonite, mixed fermentation bacterium solution are pressed
According to the weight ratio mixing of 1:2:1:8,50 DEG C of drying machine dryings are placed in, crushes, composite bacteria preparation is made.
The preparation method of 4 composite bacteria preparation of embodiment
The preparation method of the composite bacteria preparation of the present embodiment the following steps are included:
(1) actication of culture and purifying: by white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis point
On 2216E solid medium after not being inoculated into improvement, cultivated 22 hours in 28 DEG C；2216E solid culture after the improvement
The culture medium prescription of base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL, agar 25.0g and liquid microelement
1ml, the Trace Elements of the liquid microelement are as follows: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O,
The MnSO of 0.5g/L4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4, adjust pH and sterilize for 7.6,121 DEG C, 101KPa
30min；Picking single colonie is inoculated into 2216E solid slope culture medium, cultivates 20 hours in 28 DEG C of purifying, resulting test tube slant
Bacterial strain is Purify bacterial strain, and the 2216E solid slope culture medium ingredient is the same as the 2216E solid medium after improvement；
(2) it makes triangular flask liquid spawn: test tube slant bacterial strain is inoculated in the 2216E fluid nutrient medium after improvement respectively
On, it is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask liquid spawn of each bacterial strain；2216E liquid training after the improvement
The culture medium prescription for supporting base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL and liquid microelement 1ml, described
The trace element suite of liquid microelement is divided into: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O, 0.5g/L's
MnSO4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4；
(4) preparation of mixed bacteria liquid: by the triangular flask liquid spawn of cultured each bacterial strain according to white tail feather bacillus
BBHS-01, bacillus subtilis and bacillus licheniformis are inoculated into 2216E fluid nutrient medium with the bacterial population ratio of 8:1:1,
It is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask mixing liquid strain of three bacterial strains；
(5) preparation of composite bacteria preparation:
A. yeast powder, peptone, sodium chloride, bean cake powder, wheat bran skin and water are mixed with the weight ratio of 2:6:1:1:2:10
Uniformly, culture medium solid is made；
B. zeolite powder and medical stone powder are crushed respectively, and sieved with 100 mesh sieve, by zeolite powder, peanut meal and medical stone powder with
The weight ratio of 1:1:1 mixes, and gained mixture is as auxiliary material filler；
C. ooze is crushed with pulverizer, is sieved with 100 mesh sieve, is added in extractor after sieving, be 50% by mass concentration
Alcohol is added in extractor, and alcohol dosage is 10 times of ooze weight, opens stirring, extractor is placed in water bath with thermostatic control shaking table,
30min is extracted under the conditions of 140rpm, 70~80 DEG C, 100 mesh filter to take filtrate, and filter residue, which returns, to be repeated to extract one in extractor
Secondary, 100 mesh filter to take filtrate, merge gained filtrate twice, obtain ooze leachate；
D. weigh following each component according to following parts by weight: wood fragments consider 14 parts, 10 parts of wheat straw, 10 parts of mulberry leaf, duckweed 9 to be worth doing
Part, 9 parts of david poplar bark, 5 parts of fish scale, 4 parts of balsam pear, 1 part of reed root, 1 part of distiller's dried grain, 1 part of dry ferment, 1 part of conch meal, will weigh
Each component mix, and into mixture be added equivalent purified water, stir and evenly mix, be put into glass container, 28 DEG C~30 DEG C items
It is protected from light fermentation 56 hours under part, obtains tunning, by 100 mesh net filtrations of gained tunning, takes the resulting fermentation of filtering
Liquid；
E. culture medium solid, ooze leachate, triangular flask mixing liquid strain is equal according to the weight ratio of 4:1:60
Even mixing is subsequently placed in fermentor in 28 DEG C, 180~200rpm of mixing speed, ventilatory capacity 4V/V.min, cultivates 5d, obtain
To mixed fermentation bacterium solution；
F., bentonite is crushed to and is crossed 150 meshes, by auxiliary material filler, fermentation liquid, bentonite, mixed fermentation bacterium solution according to
The weight ratio of 1:1:1:9 mixes, and is placed in 50 DEG C of drying machine dryings, crushes, composite bacteria preparation is made.
The preparation method of 5 composite bacteria preparation of embodiment
The preparation method of the composite bacteria preparation of the present embodiment the following steps are included:
(1) actication of culture and purifying: by white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis point
On 2216E solid medium after not being inoculated into improvement, cultivated 22 hours in 28 DEG C；2216E solid culture after the improvement
The culture medium prescription of base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL, agar 25.0g and liquid microelement
1ml, the Trace Elements of the liquid microelement are as follows: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O,
The MnSO of 0.5g/L4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4, adjust pH and sterilize for 7.6,121 DEG C, 101KPa
30min；Picking single colonie is inoculated into 2216E solid slope culture medium, cultivates 20 hours in 28 DEG C of purifying, resulting test tube slant
Bacterial strain is Purify bacterial strain, and the 2216E solid slope culture medium ingredient is the same as the 2216E solid medium after improvement；
(2) it makes triangular flask liquid spawn: test tube slant bacterial strain is inoculated in the 2216E fluid nutrient medium after improvement respectively
On, it is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask liquid spawn of each bacterial strain；2216E liquid training after the improvement
The culture medium prescription for supporting base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL and liquid microelement 1ml, described
The trace element suite of liquid microelement is divided into: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O, 0.5g/L's
MnSO4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4；
(4) preparation of mixed bacteria liquid: by the triangular flask liquid spawn of cultured each bacterial strain according to white tail feather bacillus
BBHS-01, bacillus subtilis and bacillus licheniformis are inoculated into 2216E fluid nutrient medium with the bacterial population ratio of 6:1:1,
It is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask mixing liquid strain of three bacterial strains；
(5) preparation of composite bacteria preparation:
A. yeast powder, peptone, sodium chloride, bean cake powder, wheat bran skin and water are mixed with the weight ratio of 2:6:1:1:2:10
Uniformly, culture medium solid is made；
B. zeolite powder, peanut meal and medical stone powder are mixed with the weight ratio of 1:1:1, gained mixture is as auxiliary material
Filler；
C. ooze is crushed with pulverizer, is sieved with 100 mesh sieve, is added in extractor after sieving, be 50% by mass concentration
Alcohol is added in extractor, and alcohol dosage is 10 times of ooze weight, opens stirring, extractor is placed in water bath with thermostatic control shaking table,
30min is extracted under the conditions of 140rpm, 70~80 DEG C, 100 mesh filter to take filtrate, and filter residue, which returns, to be repeated to extract one in extractor
Secondary, 100 mesh filter to take filtrate, merge gained filtrate twice, obtain ooze leachate；
D. weigh following each component according to following parts by weight: wood fragments consider 12 parts, 12 parts of wheat straw, 12 parts of mulberry leaf, duckweed 10 to be worth doing
Part, 10 parts of david poplar bark, 5 parts of fish scale, 5 parts of balsam pear, 1 part of reed root, 1 part of distiller's dried grain, 1 part of dry ferment, 1 part of conch meal, will weigh
Good each component mixes, and the purified water of equivalent is added into mixture, stirs and evenly mixs, and is put into glass container, and 28 DEG C~30 DEG C
Under the conditions of be protected from light fermentation 56 hours, obtain tunning, by 100 mesh net filtrations of gained tunning, take and filter resulting hair
Zymotic fluid；
E. culture medium solid, ooze leachate, triangular flask mixing liquid strain is equal according to the weight ratio of 4:1:60
Even mixing is subsequently placed in fermentor in 28 DEG C, 180~200rpm of mixing speed, ventilatory capacity 4V/V.min, cultivates 5d, obtain
To mixed fermentation bacterium solution；
F. auxiliary material filler, fermentation liquid, bentonite, mixed fermentation bacterium solution are mixed according to the weight ratio of 1:2:1:9, is placed in
50 DEG C of drying machine dryings crush, composite bacteria preparation are made.
Degradation experiment of the resulting composite bacteria preparation of 6 3~embodiment of embodiment of embodiment 5 to water pollutant
1, composite bacteria preparation tests the degradation effect of prawn feed leachate
Experiment degradation solution used: being ground into fine powder for commercially available prawn feed 20g, and sterilize Chen Haishui 1000ml, after impregnating 48h
Centrifuging and taking supernatant, adds sodium nitrite 0.058g, adjusts pH 7.8, is distributed into 100mL/ bottles, in 121 DEG C, high pressure sterilization 20min.
The resulting composite bacteria preparation 1mg of 3~embodiment of Example 5 respectively is respectively added to the prawn feed drop of 100mL
It solves in liquid, 28 ± 1 DEG C, shake culture 5d under 160r/min, daily timing sampling measure the ammonia nitrogen (NH in sample liquid4-N)、
Cultured water (NO2- N) and nitrate nitrogen (NO3- N) content, thalli growth amount uses photoelectric turbidimetry with OD600It indicates, bacterium is not added
The conduct control treatment of preparation, each processing set 3 repetitions.
2, experimental result
Experimental result is shown in Table 3~table 6 respectively.The results showed that composite bacteria preparation is to prawn feed degradation solution with bright
Aobvious degradation effect, during the experiment with the growth of bacterium, inorganic nitrogen component in the bait degradation solution that can significantly degrade, wherein
To NH4-N、NO2- N and NO3The highest degradation rate of-N is respectively 93.27%, 100.00% and 92.09%, composite bacteria preparation pair
The degradation effect of pollutant is better than single strain.
The resulting composite bacteria preparation of 3 embodiment of table 3 is in different incubation times to the degradation effect (1~5d) of bait degradation solution
The resulting composite bacteria preparation of 4 embodiment of table 3 is in different incubation times to the degradation effect (1~5d) of bait degradation solution
The resulting composite bacteria preparation of 5 embodiment of table 4 is in different incubation times to the degradation effect (1~5d) of bait degradation solution
The resulting composite bacteria preparation of 6 embodiment of table 5 is in different incubation times to the degradation effect (1~5d) of bait degradation solution
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, without departing from the principle of the present invention, several improvement can also be made, these improvement should be regarded as guarantor of the invention
Protect range.
Claims (8)
1. the bacillus of a high-efficiency degradation nitrogen pollutant is white tail feather bacillus BBHS-01 (Bacillus
Baekryungensis), on December 7th, 2012 in China Committee for Culture Collection of Microorganisms's common micro-organisms preservation
The heart has carried out preservation, deposit number are as follows: CGMCC NO.6939.
2. a kind of bacillus BBHS-01 of efficient degradation nitrogen pollutant described in claim 1 is dirty in degradation aquaculture nitrogen
Contaminate the application in object.
3. one kind is with bacillus BBHS-01 composite bacteria preparation as main component described in claim 1, it is characterised in that:
The composite bacteria preparation is by white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis with 6~8:1:1
Bacterial population ratio is combined.
4. a kind of preparation method as claimed in claim 3 with bacillus BBHS-01 composite bacteria preparation as main component, institute
State the preparation method of composite bacteria preparation the following steps are included:
(1) actication of culture and purifying: white tail feather bacillus BBHS-01, bacillus subtilis and bacillus licheniformis are connect respectively
In kind to the 2216E solid medium after improvement, cultivated 22 hours in 28 DEG C；2216E solid medium after the improvement
It is formulated as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL, agar 25g and liquid microelement 1ml, it is described micro
The Trace Elements of element liquid are as follows: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2The MnSO of O, 0.5g/L4,
The FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4, pH is adjusted as 7.6,121 DEG C, 101KPa sterilizing 30min；Picking single colonie
It is inoculated into 2216E solid slope culture medium, is cultivated 20 hours in 28 DEG C of purifying, resulting test tube slant bacterial strain is Purify bacterium
Strain, the 2216E solid slope culture medium ingredient is the same as the 2216E solid medium after improvement；
(2) triangular flask liquid spawn is made: on the 2216E fluid nutrient medium after test tube slant bacterial strain to be inoculated in improvement respectively,
It is cultivated 20 hours in 28 DEG C, 160rpm, obtains the triangular flask liquid spawn of each bacterial strain；2216E Liquid Culture after the improvement
The formula of base is as follows: peptone 6.0g, yeast powder 3.0g, Chen Haishui 1000mL, agar 25g and liquid microelement 1ml, described
The trace element suite of liquid microelement is divided into: the K of 0.4g/L2HPO4·3H2The MgSO of O, 0.3g/L4·7H2O, 0.5g/L's
MnSO4, the FeSO of 0.1g/L4·7H2The CuSO of O, 0.2g/L4；
(4) preparation of mixed bacteria liquid: by the triangular flask liquid spawn of cultured each bacterial strain according to white tail feather bacillus BBHS-
01, bacillus subtilis and bacillus licheniformis are inoculated into 2216E fluid nutrient medium with the bacterial population ratio of 6~8:1:1, in
28 DEG C, 160rpm culture 20 hours, obtain the triangular flask mixing liquid strain of three bacterial strains；
(5) preparation of composite bacteria preparation:
A. yeast powder, peptone, sodium chloride, bean cake powder, wheat bran skin and water are uniformly mixed with the weight ratio of 2:6:1:1:2:10,
Culture medium solid is made；
B. zeolite powder, peanut meal and medical stone powder are mixed with the weight ratio of 1:1:1, gained mixture is filled as auxiliary material
Object；
C. ooze is crushed with pulverizer, is sieved with 100 mesh sieve, is added in extractor after sieving, the alcohol for being 50% by mass concentration
It being added in extractor, alcohol dosage is 10 times of ooze weight, stirring opened, extractor is placed in water bath with thermostatic control shaking table,
140rpm, extract 30min under the conditions of 70~80 DEG C, 100 mesh filter to take filtrate, filter residue return repeat to extract in extractor it is primary,
100 mesh filter to take filtrate, merge gained filtrate twice, obtain ooze leachate；
D. weigh each component of following parts by weight: wood fragments consider 12~15 parts, 10~12 parts of wheat straw, 10~12 parts of mulberry leaf, duckweed 9 to be worth doing
~10 parts, 9~10 parts of david poplar bark, 5 parts of fish scale, 4~5 parts of balsam pear, 1 part of reed root, 1 part of distiller's dried grain, 1 part of dry ferment, conch meal 1
Part, load weighted each component is mixed, and the purified water of equivalent is added into mixture, stirs and evenly mixs, be put into glass container, 28
DEG C~30 DEG C under the conditions of be protected from light fermentation 56 hours, obtain tunning, by 100 mesh net filtrations of gained tunning, take filtering
Resulting fermentation liquid；
E. culture medium solid, ooze leachate, triangular flask mixing liquid strain are uniformly mixed according to the weight ratio of 4:1:60
It closes, is subsequently placed in fermentor in 28 DEG C, 180~200rpm of mixing speed, ventilatory capacity 4V/V.min, cultivates 5d, mixed
Close zymocyte liquid；
F. auxiliary material filler, fermentation liquid, bentonite, mixed fermentation bacterium solution are mixed according to the weight ratio of 1:1~2:1:8~9, is set
In 50 DEG C of drying machine dryings, crushes, composite bacteria preparation is made.
5. the preparation method of composite bacteria preparation according to claim 4, it is characterised in that: the zeolite powder and medical stone powder
Partial size be 100 mesh, the bentonitic partial size be 150 mesh.
6. the preparation method of composite bacteria preparation according to claim 4, it is characterised in that: in step (4), white tail feather gemma bar
Bacterium BBHS-01, bacillus subtilis and bacillus licheniformis are inoculated into 2216E fluid nutrient medium with the bacterial population ratio of 7:1:1
In.
7. the preparation method of composite bacteria preparation according to claim 4, it is characterised in that: the d operating procedure of step (5)
In, the parts by weight of weighed each component are as follows: wood fragments consider 15 parts, 12 parts of wheat straw, 10 parts of mulberry leaf, 10 parts of duckweed, david poplar bark 9 to be worth doing
Part, 5 parts of fish scale, 4 parts of balsam pear, 1 part of reed root, 1 part of distiller's dried grain, 1 part of dry ferment, 1 part of conch meal.
8. the preparation method of the composite bacteria preparation according to any one of claim 4-7, it is characterised in that: the f of step (5)
In operating procedure, auxiliary material filler, fermentation liquid, bentonite mixed fermentation bacterium solution are mixed according to the weight ratio of 1:2:1:8.
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