CN104694412A - Bacillus cereus as well as preparation and application thereof - Google Patents
Bacillus cereus as well as preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus cereus as well as, a screening method, a preparation and application thereof. The bacillus cereus has efficient oxytetracycline degrading capability, and is screened according to the following method: (1) selecting pig manure bacterial suspension contaminated by oxytetracycline, and culturing single bacterial colonies in a culture medium containing 400-500mug/ml of cycloheximide; (2) continually improving the concentration of the oxytetracycline in the culture medium, to retain adaptive single bacterial colonies; and (3) culturing the single bacterial colonies in an inorganic salt culture medium, continually improving the concentration of the oxytetracycline, and preserving bacteria of which the oxytetracycline degrading rate is not less than 50% and the OD600 is not less than 0.2. The preparation comprises dried bacillus cereus powder and organic matrix in a mass ratio of 1:4 to 1:5. The bacillus cereus is applied to pig manure composting, has strong adaptability, high oxytetracycline degrading rate, low environmental influence and low cost, and is suitable for large-scale application.
Description
Technical field
The invention belongs to microbial technology field, more specifically, relate to a kind of bacillus cereus, its screening method, preparation and application.
Background technology
Microbiotic is widely used in China's livestock breed aquatics, and with its body or transformation form along with animal excrement and urine enter environment, and accumulate in the environment, cause microbiotic in environment to pollute.Original microbe species balance in meeting welding after microbiotic entered environment, thus cause the corresponding ecosystem unbalance.In addition, the terramycin in environment can also enter human body by biologic chain, threatens to HUMAN HEALTH.For this reason, the Department of Science and Technology of China starts national high-tech research evolutionary operation(EVOP) (863 Program), sets up " intensive cultivation exogenous chemicals Pollution control technology " key special subjects, and the microbiotic caused mainly for intensive cultivation field pollutes from source enterprising row relax.Terramycin, as the maximum veterinary antibiotic of current usage quantity, becomes the key problem of tackling of key scientific and technical problems.
In recent years, residual antibiotic degraded in the environment, conversion, move, return with a lot and in rising trend on the relevant research of the impact such as animals and plants, microorganism, become domestic and international study hotspot, but about the research of how to cut down residual antibiotic in environment at present also more rare.
Current microbiotic Pollution control technology mainly comprises physics, chemistry and biologic treating technique.Antibiotic physical removal methods mainly contains the methods such as charcoal absorption, colloid absorption, flocculation sediment and membrane filtration.And antibiotic chemical minimizing technology is now based on ion method.Due to antibiotic biological degradation other physics, chemical process relatively, have with low cost, that environmental influence is less unique advantage, at present to be subject to extensive concern, and microorganism effect wherein have also been obtained primary study.Xu Xiaolings etc. screen tsiklomitsin degradation bacteria strains two strain from long-term stacking the soil of the tsiklomitsin dregs of a decoction, are respectively defect shortwave Zymomonas mobilis and human pallid bacillus.They all can utilize tsiklomitsin as carbon source for growth.Li Li etc. finally find a kind of degradation rate to chlorimuronethyl 10mg/L to be 94.78% bacterium by research.
Terramycin is as the most widely used microbiotic, and its Pollution control technology is mainly compost at present, and corresponding terramycin degradation rate is only about 40%.Therefore, in the urgent need to finding a kind of compost to strengthen means to improve terramycin degradation rate.
Summary of the invention
For above defect or the Improvement requirement of prior art, the invention provides a kind of terramycin efficient degrading bacteria, its object is to terramycin of degrading, solve the common technical problem not adding bacterium composting process degraded terramycin inefficiency thus.
For achieving the above object, according to one aspect of the present invention, provide a kind of bacillus cereus of terramycin of can degrading, its Classification And Nomenclature is bacillus cereus 0214 Bacillus cereus 0214, China typical culture collection center CCTCC is preserved on March 5th, 2014, deposit number is CCTCC M 2014061, preservation ground Wuhan, China.
According to another aspect of the present invention, provide a kind of method of screening terramycin efficient degrading bacteria, comprise the following steps:
(1) single bacterium colony is cultivated: get the pig manure polluted by terramycin and be prepared into bacteria suspension, joined by bacteria suspension in basic medium, turns out the single bacterium colony of bacterium; Described basic medium contains the cycloheximide of 400 μ g/ml to 500 μ g/ml;
(2) terramycin screening: by the single bacterium colony of bacterium obtained in step (1), add the basic medium containing terramycin respectively, improve constantly the concentration of terramycin in basic medium until be not less than 200mg/L, retain the single bacterium colony of the bacterium that can adapt to described terramycin concentration;
(3) terramycin bacterial isolation: by the single bacterium colony of bacterium obtained in step (2), add minimal medium respectively to cultivate, improve constantly terramycin concentration until be not less than 200mg/L, measure terramycin content and OD600 in substratum at times, calculate terramycin degradation rate, preservation terramycin degradation rate be not less than 50% and OD600 be not less than 0.2 bacterium;
Described minimal medium often rises and contains: NaH
2pO
40.05% ~ 0.1%, K
2hPO
40.05% ~ 0.1%, (NH
4)
2sO
40.1% ~ 0.2%, pH value is between 7.0 to 7.4.
According to another aspect of the present invention, provide a kind of bacillus cereus preparation, comprise described bacillus cereus dry powder and organic substrate, described genus bacillus dry powder and organic substrate mass ratio are between 1:4 to 1:5.
Preferably, described bacillus cereus preparation, is characterized in that, described organic substrate is one or more in humic acid, Semen Maydis powder, starch, wheat bran; Preferred humic acid.
According to another aspect of the present invention, provide a kind of preparation method of described bacillus cereus preparation, comprise the following steps:
A () cultivates in bacillus cereus seed liquor access fermentation tank culture medium as claimed in claim 1, inoculum size is 2%-5%, until form gemma to be not less than 90%, obtains fermented liquid; The configuration of described fermentation tank culture medium is as follows: soyflour 6 ~ 7%, Semen Maydis powder 7 ~ 8%, wheat bran 1% ~ 2%, humic acid 0.5% ~ 1%, NaH
2pO
40.05% ~ 0.1%, K
2hPO
40.05% ~ 0.1% and (NH
4)
2sO
40.1% ~ 0.2%, adjust pH is 7.0 ~ 7.4, autoclave sterilization 15-30min.
B (), by centrifugal for the fermented liquid obtained in step (a), removes supernatant liquor, add organic substrate, obtains the mixture containing bacillus cereus;
C () is dry at 30 DEG C to 50 DEG C by the mixture in step (b), control its moisture between 5% to 8%, namely obtain described bacillus cereus preparation after pulverizing.
Preferably, the preparation method of described bacillus cereus preparation, its bacillus cereus seed liquor mode of described bacillus cereus bacterial classification by multistage cultivation obtains.
Bacillus cereus provided by the invention is applied to the pig manure that terramycin pollutes.
Preferably, bacillus cereus provided by the invention is applied to the pig manure that terramycin pollutes, and comprises the following steps:
The pig manure that A, the mycin that fetches earth pollute, adds carbon source and water, and control carbon-nitrogen ratio between 20 to 25, water ratio, between 60% to 70%, adds bacillus cereus as claimed in claim 1 or its active ingredient, makes pig manure be mixed with viable bacteria number and is not less than 10
6/ g;
B, by the pig manure through processing of step A, compost under the condition of 25 DEG C to 35 DEG C, is no less than 20 days.
In general, the above technical scheme conceived by the present invention compared with prior art, can obtain following beneficial effect:
(1) the present invention selects terramycin to pollute the screening study of pig manure for terramycin efficient degrading bacteria, be based on sieve bacterial strain is indigenous bacterium in pig manure, bacterium the dregs of a decoction or soil is produced from terramycin compared to prior art, to pig manure growing environment, there is stronger adaptability, can shorten and follow-uply add the lag period of carrying out compost treatment in pig manure, play a role faster, shorten the degraded terramycin time, be applicable to large-scale application, this is significant to engineer applied;
(2) screening of terramycin efficient degrading bacteria provided by the invention, by selecting antibacterials kind and consumption, controls bacterial colony and fungal colony growth situation, thus accurate select target bacterial classification, prevent miscellaneous bacteria from disturbing, efficiency of selection is high;
(3) bacillus cereus preparation provided by the invention, by selecting suitable organic substrate, the proportioning suitable with bacillus cereus is combined, and makes bacillus cereus more be easy to preservation, and grow more rapid under being applicable to environment, degradation efficiency is higher;
(4) the present invention utilizes described bacillus cereus to carry out compost, and microbiotic of effectively degrading, relative to other physics, chemical process, with low cost, environmental influence is little.
Accompanying drawing explanation
Fig. 1 is the morphological specificity of bacillus cereus B.cereus0214;
Fig. 2 is embodiment 8 pig manure analysis of experimental data figure;
Fig. 3 is embodiment 9 pig manure analysis of experimental data figure;
Fig. 4 is embodiment 10 pig manure analysis of experimental data figure;
Fig. 5 is embodiment 11 pig manure analysis of experimental data figure.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.In addition, if below in described each embodiment of the present invention involved technical characteristic do not form conflict each other and just can mutually combine.
Bacillus cereus provided by the invention, is characterized in that, its Classification And Nomenclature is B.cereus0214, and be preserved in China typical culture collection center CCTCC on March 3rd, 2014, preservation address Wuhan, China university, deposit number is CCTCC M2014061.
Through qualification, this bacterium has following characteristics:
(1) colonial morphology: bacterium colony subcircular, canescence, opaque, bacterium colony is comparatively large, and diameter is 5 ~ 7mm, and edge is irregular in expansion shape, and surface slightly gloss is Chinese wax shape, does not secrete pigment;
(2) cellular form: Gram-positive bacillus, shaft-like, bacterial strain width is 1.0 ~ 1.2 μm, and length is 3.0 ~ 5.0 μm, atrichia, central spore, and sporangiocyst is without obviously expanding;
(3) 16S rRNA qualification result: bacillus;
(4) physiological and biochemical property: amphimicrobian, phenol red dextrose bouillon identification experiment result is positive, improvement v-p Medium on Identification experimental result is positive, nitrate broth identification experiment result is positive, power-nitrate culture-medium (A method) identification experiment result is positive, wood sugar-gelatine culture identification experiment is that color is constant, liquefy gelatin.
The invention provides a kind of screening method of terramycin efficient degrading bacteria, comprise the following steps:
(1) single bacterium colony is cultivated:
Get the pig manure polluted by terramycin and be prepared into bacteria suspension, bacteria suspension is joined in basic medium.Be inverted after culture medium solidifying culture dish cultivate in 35 DEG C of constant incubators to choose after 3 days different shape, bacterium colony clearly colonies typical cultivate, turn out the single bacterium colony of bacterium.
Described basic medium often rises and contains: peptone 1.0% ~ 1.2%, beef extract 0.3% ~ 0.5%, sodium-chlor 0.5% ~ 0.6%, agar 1.5% ~ 2.0%.
Its preparation method is: take peptone 10.0 ~ 12.0g, beef extract 3.0 ~ 5.0g, sodium-chlor 5.0 ~ 6.0g, agar 15.0 ~ 20.0g, and heating for dissolving is in 1L distilled water, and adjust pH is 7.0 ~ 7.4, autoclave sterilization 15 ~ 30min; After substratum is cooled to 48 ~ 50 DEG C, adds cycloheximide degerming after filtration, makes the concentration of cycloheximide be 400 μ g/ml to 500 μ g/ml; In described substratum, contained cycloheximide act as Antifungi growth.The method of turning out the single bacterium colony of bacterium can adopt four rides to cultivate.
(2) terramycin screening:
By the single bacterium colony of bacterium obtained in step (1), add the basic medium containing terramycin respectively, improve constantly the concentration of terramycin in basic medium until be not less than 200mg/L, retain the single bacterium colony of the bacterium that can adapt to described terramycin concentration.
(3) terramycin bacterial isolation:
By the single bacterium colony of bacterium obtained in step (2), add minimal medium respectively to cultivate, improve constantly terramycin concentration until 200mg/L, measure terramycin content and OD600 in substratum at times, calculate terramycin degradation rate, preservation terramycin degradation rate be not less than 50% and OD600 be not less than 0.2 bacterium;
Described minimal medium often rises containing NaH
2pO
40.05% ~ 0.1%, K
2hPO
40.05% ~ 0.1% and (NH
4)
2sO
40.1% ~ 0.2%.
Its preparation method is as follows: often take 0.5 ~ 1.0g NaH
2pO
4, 0.5 ~ 1gK
2hPO
4with 1.0 ~ 2.0g (NH
4)
2sO
4, slowly add ultrapure water, add ultrapure water again after fully dissolving and be settled to 1L, adjust pH between 7.0 to 7.4 at autoclave sterilization 15 ~ 30min; Now with the current.
In described minimal medium, terramycin concentration improves constantly, and improves 50mg/L at every turn; Shake flask fermentation; Adopt determined by ultraviolet spectrophotometry terramycin content and OD600, measurement period is respectively: latter 1st, 2,3 day of fermentation.
Bacillus cereus preparation provided by the invention, comprises described bacillus cereus dry powder and organic substrate, and described bacillus cereus dry powder and organic substrate mass ratio are between 1:4 ~ 1:5.Described organic substrate is one or more in humic acid, Semen Maydis powder, starch, wheat bran; Preferred humic acid.
The preparation method of bacillus cereus preparation provided by the invention, comprises the following steps:
A () cultivates in described bacillus cereus seed liquor access fermentation tank culture medium, inoculum size 2% ~ 5%, until form gemma to be not less than 90%, obtains fermented liquid.
The configuration of described fermentation tank culture medium is as follows: soyflour 6 ~ 7%, Semen Maydis powder 7 ~ 8%, wheat bran 1% ~ 2%, humic acid 0.5% ~ 1%, NaH
2pO
40.05% ~ 0.1%, K
2hPO
40.05% ~ 0.1%, (NH
4)
2sO
40.1% ~ 0.2%, adjust pH is 7.0 ~ 7.4, autoclave sterilization 15 ~ 30min.
The described bacillus cereus seed liquor mode of described bacillus cereus bacterial classification by multistage cultivation obtains, and concrete mode is as follows:
(I) first order seed: by strain inoculation in 500mL triangular flask, liquid amount is 150mL, and inoculum size is 4 ~ 5%, 35 ~ 37 DEG C, and 12 ~ 16h cultivated by 120 ~ 180rpm shaking table, obtains first order seed;
(II) secondary seed: first order seed is inoculated in 5L triangular flask, liquid amount is 2L, and inoculum size is 3 ~ 4%, 35 ~ 37 DEG C, and 12 ~ 16h cultivated by 120 ~ 180rpm shaking table, obtains secondary seed;
(III) three grade of seed: be inoculated in by secondary seed in 100L fermentor tank, liquid amount is 40L, and inoculum size is 2 ~ 3%, 35 ~ 37 DEG C, and 12 ~ 16h cultivated by 120 ~ 180rpm shaking table, obtains three grades of seeds.
The configuration of described seed culture medium is as follows: peptone 1.0% ~ 1.2%, beef extract 0.3% ~ 0.5%, sodium-chlor 0.5% ~ 0.6%, agar 1.5% ~ 2.0% maltose 0.5% ~ 1%, yeast extract 0.5% ~ 1%, NaH
2pO
40.05% ~ 0.1%, K
2hPO
40.05% ~ 0.1% and (NH
4)
2sO
40.1% ~ 0.2%, adjust pH is 7.0 ~ 7.4, autoclave sterilization 15 ~ 30min.
B (), by centrifugal for the fermented liquid obtained in step (a), removes supernatant liquor, add organic substrate, obtains the mixture containing bacillus cereus; Described organic substrate is one or more in humic acid, Semen Maydis powder, starch, wheat bran; Preferred humic acid.
(c) by the mixture in step (b) at 30 DEG C to 50 DEG C, preferably dry at 40 DEG C, control its moisture 5% ~ 8%, pulverize and namely obtain described bacillus cereus preparation.
Bacillus cereus provided by the invention can be applicable to the pig manure heap that terramycin pollutes, and concrete grammar is as follows:
The pig manure that A, the mycin that fetches earth pollute, adds carbon source and water, and control carbon-nitrogen ratio between 20 to 25, water ratio is between 60% to 70%, and the bacillus cereus described in interpolation or its preparation, make to be mixed with viable bacteria number in pig manure and be not less than 10
6/ g.
B, by the pig manure through processing of step A, compost under the condition of 25 DEG C to 35 DEG C, is no less than 20 days.
Preferably, described steps A adds bacillus cereus preparation provided by the invention.Bacillus cereus preparation provided by the invention is used for pig manure advantage and comprises: do not need activation before 1. using, and adds conveniently; 2. said preparation is solid-state, stable in properties, conveniently transports and stores 3. said preparation active bacteria and come from pig manure, and when using it to carry out pig manure, the adaptive phase is short, can effectively reduce the compost time; 4. test shows, is equal to the experimental group being even better than adding viable bacteria when said preparation is used for pig manure to the degradation effect of terramycin.
To sum up, this bacillus cereus preparation has good future in engineering applications.
Be below embodiment:
Embodiment 1
A screening method for terramycin efficient degrading bacteria, comprises the following steps:
(1) single bacterium colony is cultivated:
Get the pig manure polluted by terramycin and be prepared into bacteria suspension, bacteria suspension is joined in basic medium.Be inverted after culture medium solidifying culture dish cultivate in 35 DEG C of constant incubators to choose after 3 days different shape, bacterium colony clearly colonies typical cultivate, turn out the single bacterium colony of bacterium.
Described basic medium is prepared as follows: accurately take peptone 10.0g, beef extract 3.0g, sodium-chlor 5.0g, agar 15g, and heating for dissolving is in 1L distilled water, and adjust pH is 7.0, autoclave sterilization 15min; After substratum is cooled to 48 DEG C, adds cycloheximide degerming after filtration, makes the concentration of cycloheximide be 400 μ g/ml.The method of turning out the single bacterium colony of bacterium adopts four rides to cultivate.
(2) terramycin screening:
By the single bacterium colony of bacterium obtained in step (1), add the basic medium containing terramycin respectively, improve constantly the concentration of terramycin in basic medium until 200mg/L, retain the single bacterium colony of the bacterium that can adapt to described terramycin concentration.
(3) terramycin bacterial isolation:
By the single bacterium colony of bacterium obtained in step (2), add minimal medium respectively to cultivate, improve constantly terramycin concentration until 200mg/L, measure terramycin content and OD600 in substratum at times, calculate terramycin degradation rate, preservation terramycin degradation rate be not less than 50% and OD600 be not less than 0.2 bacterium;
Described minimal medium its preparation method is as follows: accurately take 0.5g NaH
2pO
4, 0.5gK
2hPO
4with 1.0g (NH
4)
2sO
4, slowly add ultrapure water, add ultrapure water again after fully dissolving and be settled to 1L, adjust pH to be 7.0, autoclave sterilization 15min; Now with the current.
In described minimal medium, terramycin concentration is followed successively by: 50mg/L, 100mg/L, 150mg/L and 200mg/L; Shake flask fermentation; Adopt determined by ultraviolet spectrophotometry terramycin content and OD600, measurement period is respectively: latter 1st, 2,3 day of fermentation.
Embodiment 2
A screening method for terramycin efficient degrading bacteria, comprises the following steps:
(1) single bacterium colony is cultivated:
Get the pig manure polluted by terramycin and be prepared into bacteria suspension, bacteria suspension is joined in basic medium.Be inverted after culture medium solidifying culture dish cultivate in 35 DEG C of constant incubators to choose after 3 days different shape, bacterium colony clearly colonies typical cultivate, turn out the single bacterium colony of bacterium.
Described basic medium is prepared as follows: accurately take peptone 12.0g, beef extract 5.0g, sodium-chlor 6.0g, agar 20g, and heating for dissolving is in 1L distilled water, and adjust pH is 7.4, autoclave sterilization 30min; After substratum is cooled to 50 DEG C, adds cycloheximide degerming after filtration, makes the concentration of cycloheximide be 500 μ g/ml.The method of turning out the single bacterium colony of bacterium adopts four rides to cultivate.
(2) terramycin screening:
By the single bacterium colony of bacterium obtained in step (1), add the basic medium containing terramycin respectively, improve constantly the concentration of terramycin in basic medium until 250mg/L, retain the single bacterium colony of the bacterium that can adapt to described terramycin concentration.
(3) terramycin bacterial isolation:
By the single bacterium colony of bacterium obtained in step (2), add minimal medium respectively to cultivate, improve constantly terramycin concentration until 250mg/L, measure terramycin content and OD600 in substratum at times, calculate terramycin degradation rate, preservation terramycin degradation rate be not less than 50% and OD600 be not less than 0.2 bacterium; Described minimal medium its preparation method is as follows: accurately take 1.0g NaH
2pO
4, 1.0g K
2hPO
4with 2.0g (NH
4)
2sO
4, slowly add ultrapure water, add ultrapure water again after fully dissolving and be settled to 1L, adjust pH 7.4, at autoclave sterilization 30min; Now with the current.
In described minimal medium, terramycin concentration is followed successively by: 50mg/L, 100mg/L, 150mg/L, 200mg/L and 250mg/L; Shake flask fermentation; Adopt determined by ultraviolet spectrophotometry terramycin content and OD600, measurement period is respectively: latter 1st, 2,3 day of fermentation.
Embodiment 3
A screening method for terramycin efficient degrading bacteria, comprises the following steps:
(1) single bacterium colony is cultivated:
Get the pig manure polluted by terramycin and be prepared into bacteria suspension, bacteria suspension is joined in basic medium.Be inverted after culture medium solidifying culture dish cultivate in 35 DEG C of constant incubators to choose after 3 days different shape, bacterium colony clearly colonies typical cultivate, turn out the single bacterium colony of bacterium.
Described basic medium is prepared as follows: accurately take peptone 11.0g, beef extract 4.0g, sodium-chlor 5.5g, agar 17.5g, and heating for dissolving is in 1L distilled water, and adjust pH is 7.2, autoclave sterilization 23min; After substratum is cooled to 49 DEG C, adds cycloheximide degerming after filtration, makes the concentration of cycloheximide be 450 μ g/ml.The method of turning out the single bacterium colony of bacterium adopts four rides to cultivate.
(2) terramycin screening:
By the single bacterium colony of bacterium obtained in step (1), add the basic medium containing terramycin respectively, improve constantly the concentration of terramycin in basic medium until 300mg/L, retain the single bacterium colony of the bacterium that can adapt to described terramycin concentration.
(3) terramycin bacterial isolation:
By the single bacterium colony of bacterium obtained in step (2), add minimal medium respectively to cultivate, improve constantly terramycin concentration until 300mg/L, measure terramycin content and OD600 in substratum at times, calculate terramycin degradation rate, preservation terramycin degradation rate be not less than 50% and OD600 be not less than 0.2 bacterium;
Described minimal medium its preparation method is as follows: accurately take 0.75g NaH
2pO
4, 0.75g K
2hPO
4with 1.5g (NH
4)
2sO
4, slowly add ultrapure water, add ultrapure water again after fully dissolving and be settled to 1L, adjust pH 7.2, at autoclave sterilization 23min; Now with the current.
In described minimal medium, terramycin concentration is followed successively by: 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L and 300mg/L; Shake flask fermentation; Adopt determined by ultraviolet spectrophotometry terramycin content and OD600, measurement period is respectively: latter 1st, 2,3 day of fermentation.
Embodiment 4
To screening the bacterium obtained in embodiment 1, identify.Through qualification, this bacterium has following characteristics:
(1) colonial morphology: bacterium colony subcircular, canescence, opaque, bacterium colony is comparatively large, and diameter is 5 ~ 7mm, and edge is irregular in expansion shape, and surface slightly gloss is Chinese wax shape, does not secrete pigment;
(2) cellular form: Gram-positive bacillus, shaft-like, bacterial strain width is 1.0 ~ 1.2 μm, and length is 3.0 ~ 5.0 μm, atrichia, central spore, and sporangiocyst is without obviously expanding;
(3) 16S rRNA qualification result: bacillus.The rRNA sequence of its rrna 16S subunit is the sequence 1 in sequence table, and its comparison result is as shown in table 1.
The 16S rRNA gene sequencing BLAST result of table 1 bacillus cereus B.cereus0214
(4) physiological and biochemical property: amphimicrobian, phenol red dextrose bouillon identification experiment result is positive, improvement v-p Medium on Identification experimental result is positive, nitrate broth identification experiment result is positive, power-nitrate culture-medium (A method) identification experiment result is positive, wood sugar-gelatine culture identification experiment is that color is constant, liquefy gelatin.
To screen the bacillus cereus obtained in embodiment 1, called after B.cereus0214, be preserved in China typical culture collection center CCTCC on March 3rd, 2014, preservation address Wuhan, China university, deposit number is CCTCC M2014061.
Embodiment 5
Bacillus cereus in embodiment 1, the preparation method of its preparation, comprises the following steps:
A () cultivates in described bacillus cereus seed liquor access fermentation tank culture medium, inoculum size 2%, until form gemma to be not less than 90%, obtains fermented liquid.
The configuration of described fermentation tank culture medium is as follows: soyflour 6%, Semen Maydis powder 7%, wheat bran 1%, humic acid 0.5%, NaH
2pO
40.05%, K
2hPO
40.05%, (NH
4)
2sO
40.1%, adjust pH is 7.0, autoclave sterilization 15min.
The described bacillus cereus seed liquor mode of described bacillus cereus bacterial classification by multistage cultivation obtains, and concrete mode is as follows:
(I) first order seed: by strain inoculation in 500mL triangular flask, liquid amount is 150mL, and inoculum size is 4%, 35 DEG C, and 12h cultivated by 120rpm shaking table, obtains first order seed;
(II) secondary seed: first order seed is inoculated in 5L triangular flask, liquid amount is 2L, and inoculum size is 3%, 35 DEG C, and 12h cultivated by 120rpm shaking table, obtains secondary seed;
(III) three grade of seed: be inoculated in by secondary seed in 100L fermentor tank, liquid amount is 40L, and inoculum size is 2%, 35 DEG C, and 12h cultivated by 120rpm shaking table, obtains three grades of seeds.
The configuration of described seed culture medium is as follows: peptone 1.0%, beef extract 0.3%, sodium-chlor 0.5%, maltose 0.5%, yeast extract 0.5%, 0.05%NaH
2pO4,0.05%K
2hPO
4with 0.1%-(NH
4)
2sO
4, adjust pH is 7.0, autoclave sterilization 15min.
B (), by centrifugal for the fermented liquid obtained in step (a), removes supernatant liquor, add organic substrate, obtains the mixture containing bacillus cereus; Described organic substrate is humic acid.
C () is dry at 40 DEG C by the mixture in step (b), control its moisture 5% ~ 8%, pulverizes and namely obtains described bacillus cereus preparation.
Obtained bacillus cereus preparation, comprises described bacillus cereus dry powder and humic acid.
Embodiment 6
Bacillus cereus in embodiment 1, the preparation method of its preparation, comprises the following steps:
A () cultivates in described bacillus cereus seed liquor access fermentation tank culture medium, inoculum size 5%, until form gemma to be not less than 90%, obtains fermented liquid.
The configuration of described fermentation tank culture medium is as follows: soyflour 7%, Semen Maydis powder 8%, wheat bran 2%, humic acid 1%, NaH
2pO
40.1%, K
2hPO
40.1%, (NH
4)
2sO
40.2%, adjust pH is 7.4, autoclave sterilization 30min.
The described bacillus cereus seed liquor mode of described bacillus cereus bacterial classification by multistage cultivation obtains, and concrete mode is as follows:
(I) first order seed: by strain inoculation in 500mL triangular flask, liquid amount is 150mL, and inoculum size is 5%, 37 DEG C, and 16h cultivated by 180rpm shaking table, obtains first order seed;
(II) secondary seed: first order seed is inoculated in 5L triangular flask, liquid amount is 2L, and inoculum size is 4%, 37 DEG C, and 16h cultivated by 180rpm shaking table, obtains secondary seed;
(III) three grade of seed: be inoculated in by secondary seed in 100L fermentor tank, liquid amount is 40L, and inoculum size is 3%, 37 DEG C, and 16h cultivated by 180rpm shaking table, obtains three grades of seeds.
The configuration of described seed culture medium is as follows: peptone 1.2%, beef extract 0.5%, sodium-chlor 0.6%, maltose 1%, yeast extract 1%, NaH
2pO40.1%, K
2hPO
40.1% and (NH
4)
2sO
40.2%, adjust pH is 7.4, autoclave sterilization 30min.
B (), by centrifugal for the fermented liquid obtained in step (a), removes supernatant liquor, add organic substrate, obtains the mixture containing bacillus cereus; Described organic substrate is Semen Maydis powder.
C () is dry at 30 DEG C by the mixture in step (b), control its moisture 6.5%, pulverizes and namely obtains described bacillus cereus preparation.
Obtained bacillus cereus preparation, comprise described bacillus cereus dry powder and Semen Maydis powder, described bacillus cereus dry powder and Semen Maydis powder mass ratio are 1:5.
Embodiment 7
Bacillus cereus in embodiment 1, the preparation method of its preparation, comprises the following steps:
A () cultivates in described bacillus cereus seed liquor access fermentation tank culture medium, inoculum size 3.5%, until form gemma to be not less than 90%, obtains fermented liquid.
The configuration of described fermentation tank culture medium is as follows: soyflour 6.5%, Semen Maydis powder 7.5%, wheat bran 1.5%, humic acid 0.75%, NaH
2pO
40.075%, K
2hPO
40.075%, (NH
4)
2sO
40.15%, adjust pH is 7.2, autoclave sterilization 23min.
The described bacillus cereus seed liquor mode of described bacillus cereus bacterial classification by multistage cultivation obtains, and concrete mode is as follows:
(I) first order seed: by strain inoculation in 500mL triangular flask, liquid amount is 150mL, and inoculum size is 4.5%, 36 DEG C, and 14h cultivated by 150rpm shaking table, obtains first order seed;
(II) secondary seed: first order seed is inoculated in 5L triangular flask, liquid amount is 2L, and inoculum size is 3.5%, 36 DEG C, and 14h cultivated by 150rpm shaking table, obtains secondary seed;
(III) three grade of seed: be inoculated in by secondary seed in 100L fermentor tank, liquid amount is 40L, and inoculum size is 2.5%, 36 DEG C, and 14h cultivated by 150rpm shaking table, obtains three grades of seeds.
The configuration of described seed culture medium is as follows: peptone 1.1%, beef extract 0.4%, sodium-chlor 0.55%, maltose 0.75%, yeast extract 0.75%, agar 1.75%, NaH
2pO
40.075%, K
2hPO
40.075% and (NH
4)
2sO
40.15%, adjust pH is 7.2, autoclave sterilization 23min.
B (), by centrifugal for the fermented liquid obtained in step (a), removes supernatant liquor, add organic substrate, obtains the mixture containing bacillus cereus; Described organic substrate is humic acid and starch.
C () is dry at 50 DEG C by the mixture in step (b), control its moisture 6.5%, pulverizes and namely obtains described bacillus cereus preparation.
Obtained bacillus cereus preparation, comprise described bacillus cereus dry powder, humic acid and starch, described bacillus cereus dry powder, humic acid and starch quality ratio are 1:1.5:3.。
Embodiment 8
Bacillus cereus preparation obtained in embodiment 5, be applied to pig manure, concrete steps are as follows:
The pig manure that A, the mycin that fetches earth pollute, adds carbon source and water, and control carbon-nitrogen ratio 20, water ratio is 60%, the bacillus cereus preparation described in interpolation, makes to be mixed with viable bacteria number in pig manure and is being not less than 10
6/ g.
B, by the pig manure through processing of step A, compost under the condition of 25 DEG C, 21d.
Embodiment 9
Bacillus cereus preparation obtained in embodiment 5, be applied to pig manure, concrete steps are as follows:
The pig manure that A, the mycin that fetches earth pollute, adds carbon source and water, and control carbon-nitrogen ratio 25, water ratio is 70%, the bacillus cereus preparation described in interpolation, makes to be mixed with viable bacteria number in pig manure and is being not less than 10
6/ g.
B, by the pig manure through processing of step A, compost under the condition of 35 DEG C, 24d.
Embodiment 10
Bacillus cereus preparation obtained in embodiment 5, be applied to pig manure, concrete steps are as follows:
The pig manure that A, the mycin that fetches earth pollute, adds carbon source and water, and control carbon-nitrogen ratio 22.5, water ratio is 65%, the bacillus cereus preparation described in interpolation, makes to be mixed with viable bacteria number in pig manure and is being not less than 10
6/ g.
B, by the pig manure through processing of step A, compost under the condition of 30 DEG C, 27d.
Embodiment 11
Bacillus cereus obtained in embodiment 1, be applied to pig manure, concrete steps are as follows:
The pig manure that A, the mycin that fetches earth pollute, adds carbon source and water, and control carbon-nitrogen ratio 22.5, water ratio is 65%, the bacillus cereus described in interpolation, makes to be mixed with viable bacteria number in pig manure and is being not less than 106/g.
B, by the pig manure through processing of step A, compost under the condition of 30 DEG C, 21d.
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. a bacillus cereus, is characterized in that, its Classification And Nomenclature is B.cereus0214, and be preserved in China typical culture collection center CCTCC on March 3rd, 2014, deposit number is CCTCC M2014061.
2. a terramycin efficient degradation bacterial screening method, is characterized in that, comprises the following steps:
(1) single bacterium colony is cultivated: get the pig manure polluted by terramycin and be prepared into bacteria suspension, joined by bacteria suspension in basic medium, turns out the single bacterium colony of bacterium; Described basic medium contains the cycloheximide of 400 μ g/ml to 500 μ g/ml;
(2) terramycin screening: by the single bacterium colony of bacterium obtained in step (1), add the basic medium containing terramycin respectively, improve constantly the concentration of terramycin in basic medium until be not less than 200mg/L, retain the single bacterium colony of the bacterium that can adapt to described terramycin concentration;
(3) terramycin bacterial isolation: by the single bacterium colony of bacterium obtained in step (2), add minimal medium respectively to cultivate, improve constantly terramycin concentration until be not less than 200mg/L, measure terramycin content and OD600 in substratum at times, calculate terramycin degradation rate, preservation terramycin degradation rate be not less than 50% and OD600 be not less than 0.2 bacterium;
Described minimal medium often rises and contains: NaH
2pO
40.05% ~ 0.1%, K
2hPO
40.05% ~ 0.1%, (NH
4)
2sO
40.1% ~ 0.2%, pH value is between 7.0 to 7.4.
3. a bacillus cereus preparation, is characterized in that, comprises bacillus cereus dry powder as claimed in claim 1 and organic substrate, and described genus bacillus dry powder and organic substrate mass ratio are between 1:4 to 1:5.
4. bacillus cereus preparation as claimed in claim 3, is characterized in that, described organic substrate is one or more in humic acid, Semen Maydis powder, starch, wheat bran; Preferred humic acid.
5. the preparation method of the bacillus cereus preparation as described in claim 3 or 4, is characterized in that, comprise the following steps:
A () cultivates in bacillus cereus seed liquor access fermentation tank culture medium as claimed in claim 1, inoculum size is 2%-5%, until form gemma to be not less than 90%, obtains fermented liquid; The configuration of described fermentation tank culture medium is as follows: soyflour 6 ~ 7%, Semen Maydis powder 7 ~ 8%, wheat bran 1% ~ 2%, humic acid 0.5% ~ 1%, NaH
2pO
40.05% ~ 0.1%, K
2hPO
40.05% ~ 0.1% and (NH
4)
2sO
40.1% ~ 0.2%, adjust pH is 7.0 ~ 7.4, autoclave sterilization 15-30min.
B (), by centrifugal for the fermented liquid obtained in step (a), removes supernatant liquor, add organic substrate, obtains the mixture containing bacillus cereus;
C () is dry at 30 DEG C to 50 DEG C by the mixture in step (b), control its moisture between 5% to 8%, namely obtain described bacillus cereus preparation after pulverizing.
6. preparation method as claimed in claim 5, is characterized in that, the described bacillus cereus seed liquor mode of described bacillus cereus bacterial classification by multistage cultivation obtains.
7. bacillus cereus as claimed in claim 1 is applied to the pig manure that terramycin pollutes.
8. bacillus cereus as claimed in claim 7 is applied to the pig manure that terramycin pollutes, and it is characterized in that, comprises the following steps:
The pig manure that A, the mycin that fetches earth pollute, adds carbon source and water, and control carbon-nitrogen ratio between 20 to 25, water ratio, between 60% to 70%, adds bacillus cereus as claimed in claim 1 or its active ingredient, makes pig manure be mixed with viable bacteria number and is not less than 10
6/ g;
B, by the pig manure through processing of step A, compost under the condition of 25 DEG C to 35 DEG C, is no less than 20 days.
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Cited By (5)
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| CN111268805A (en) * | 2020-01-21 | 2020-06-12 | 中国科学院成都生物研究所 | Bacillus cereus and application thereof in pig manure biogas slurry flocculation |
| CN111517837A (en) * | 2020-05-21 | 2020-08-11 | 齐鲁工业大学 | Livestock and poultry manure composite sulfuric acid type furfural residue organic fertilizer and preparation method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635900A (en) * | 2016-12-15 | 2017-05-10 | 中国农业科学院农业资源与农业区划研究所 | Screening method of oxytetracycline degrading strain |
| CN106701646A (en) * | 2017-03-30 | 2017-05-24 | 广东省农业科学院农业资源与环境研究所 | Tetracycline degrading bacterium and application thereof to livestock and poultry excrement composting treatment |
| CN111268805A (en) * | 2020-01-21 | 2020-06-12 | 中国科学院成都生物研究所 | Bacillus cereus and application thereof in pig manure biogas slurry flocculation |
| CN111268805B (en) * | 2020-01-21 | 2022-02-08 | 中国科学院成都生物研究所 | Bacillus cereus and application thereof in pig manure biogas slurry flocculation |
| CN111517837A (en) * | 2020-05-21 | 2020-08-11 | 齐鲁工业大学 | Livestock and poultry manure composite sulfuric acid type furfural residue organic fertilizer and preparation method thereof |
| CN113755400A (en) * | 2021-10-11 | 2021-12-07 | 吉林农业大学 | Bacillus cereus for degrading aureomycin and application thereof |
| CN113755400B (en) * | 2021-10-11 | 2023-03-03 | 吉林农业大学 | Bacillus cereus for degrading aureomycin and application thereof |
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