CN110079481A - One bacillus amyloliquefaciens SL-7 and its application - Google Patents
One bacillus amyloliquefaciens SL-7 and its application Download PDFInfo
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Abstract
The present invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SL-7 and its applications, are related to microorganisms technical field.For bacillus amyloliquefaciens SL-7 of the present invention screened from tobacco straw waste, deposit number is GDMCC No:60630.In embodiments of the present invention, the bacillus amyloliquefaciens SL-7 can produce apparent cellulose degradation circle；In cellulase activity test, CMCase reaches as high as 84.81U/mL, and FPase reaches as high as 35.40U/mL；In phosphorus decomposing test, apparent phosphorus decomposing circle is all shown for organic phosphorus and Phos.Therefore the bacillus amyloliquefaciens SL-7 can be used for degraded cellulose and improve phosphorus content in soil, to prepare tabacco straw compost.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a bacillus amyloliquefaciens SL-7 and its application.
Background technique
China is the most country of World tobacco cultivated area, and annual yield of tobacco is about 450~5,000,000 tons, same with this
When produce largely discarded cigarette stalk.Content of organics is about 90~95% in tabacco straw, and content of mineral substances is 5~10%,
Wherein nitrogen content 0.5~1.2%, phosphorus content 0.1~-0.25%, potassium content 0.8~1.8%；There are many also containing in stalk simultaneously
Microorganism, but the microorganism screened from tabacco straw at present, mostly simple function or degraded cellulose, or degradation
Nicotine, or for producing laccase, it is not a kind of screened from tabacco straw while micro- life with multiple functions
Object.
Summary of the invention
In view of this, the purpose of the present invention is to provide a bacillus amyloliquefaciens SL-7 and its application, the Xie Dian
Afnyloliquefaciens SL-7 is screened from tobacco straw waste, and not only degraded cellulose is powerful, also has the function of phosphorus decomposing.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SL-7, preservation is compiled
Number be GDMCC No:60630.
Preferably, the 16S gene order of the bacillus amyloliquefaciens SL-7 is as shown in SEQ ID NO.1.
The present invention also provides application of the bacillus amyloliquefaciens SL-7 in degraded cellulose.
The present invention also provides application of the bacillus amyloliquefaciens SL-7 in phosphorus decomposing.
The present invention also provides the bacillus amyloliquefaciens SL-7 to prepare the application in degrading tobacco stalk microbial inoculum.
Preferably, the preparation method of the degrading tobacco stalk microbial inoculum, comprising the following steps: by the solution starch gemma bar
Bacterium SL-7 is inoculated in CMC fermentation medium and cultivates 3d, obtains degrading tobacco stalk microbial inoculum；In the CMC fermentation medium with
Peptone is only nitrogen source, CMC-Na is sole carbon source, and the C/N ratio of the sole carbon source and only nitrogen source is 2:1.
Preferably, the inoculation volume of the bacillus amyloliquefaciens SL-7 be the CMC fermentation medium volume 6~
10%.
Preferably, the temperature of the culture is 37 DEG C, and pH value is 6.0~8.0.
Preferably, with stirring in the incubation, the revolving speed of the stirring is 180~200rpm.
The present invention also provides the degrading tobacco stalk microbial inoculums to prepare the application in tabacco straw compost.
The present invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SL-7, the solutions
Bacillus amyloliquefaciens SL-7 is screened from tobacco straw waste.In embodiments of the present invention, by verification experimental verification, the solution starch
Bacillus SL-7 can produce apparent cellulose degradation circle；In cellulase activity test, CMCase reaches as high as 424.04U/
ML, FPase reach as high as 176.99U/mL；In phosphorus decomposing test, apparent phosphorus decomposing is all shown for organic phosphorus and Phos
Circle.Bacillus amyloliquefaciens SL-7 of the present invention can be used in degraded cellulose and phosphorus decomposing, to prepare tabacco straw compost.
Biological deposits information
One bacillus amyloliquefaciens (Bacillus amyloliquefaciens), biomaterial title and dated mirror
Determining feature is Bacillus amyloliquefaciens SL-7, and preservation place is Guangdong Province's Culture Collection, is protected
Hiding address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building, and the preservation time is on April 11st, 2019, and deposit number is
GDMCC No:60630。
Detailed description of the invention
Fig. 1 is hydrolysis effect figure of the SL-7 bacterial strain on Congo red culture medium；
Fig. 2 is cellulase activity measurement result figure；
Fig. 3 is dissolving P capacity measurement result figure；
Fig. 4 is that revolving speed influences SL-7 strain enzyme-producing total enzyme activity；
Fig. 5 is that cultivation temperature influences SL-7 strain enzyme-producing total enzyme activity；
Fig. 6 is that inoculation volume influences SL-7 strain enzyme-producing total enzyme activity；
Fig. 7 is that initial pH value influences SL-7 strain enzyme-producing total enzyme activity；
Fig. 8 is that different carbon source influences SL-7 strain enzyme-producing total enzyme activity；
Fig. 9 is that different nitrogen sources influence SL-7 strain enzyme-producing total enzyme activity；
Figure 10 is that different carbon-nitrogen ratios influence SL-7 strain enzyme-producing total enzyme activity.
Specific embodiment
The present invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SL-7, preservation is compiled
Number be GDMCC No:60630.The 16S gene order of bacillus amyloliquefaciens SL-7 of the present invention such as SEQ ID NO.1 institute
Show.
The present invention also provides application of the bacillus amyloliquefaciens SL-7 in degraded cellulose.
The present invention also provides application of the bacillus amyloliquefaciens SL-7 in phosphorus decomposing.
The present invention also provides the bacillus amyloliquefaciens SL-7 to prepare the application in degrading tobacco stalk microbial inoculum.
The preparation method of degrading tobacco stalk microbial inoculum of the present invention, preferably includes following steps: by the solution starch bud
Spore bacillus SL-7, which is inoculated in CMC fermentation medium, cultivates 3d, obtains degrading tobacco stalk microbial inoculum；The CMC fermentation medium
In using peptone be only nitrogen source, CMC-Na for sole carbon source, the C/N ratio of the sole carbon source and only nitrogen source is 2:1.This
The inoculation volume for inventing the bacillus amyloliquefaciens SL-7 is preferably the 6~10% of the CMC fermentation medium volume, more excellent
It is selected as 8%.The temperature of culture of the present invention is preferably 25~45 DEG C, and more preferably 37 DEG C.The pH value of culture of the present invention
Preferably 6.0~8.0, more preferably 7.0.The present invention is in the incubation preferably with stirring, the revolving speed of the stirring
Preferably 180~200rpm, more preferably 200rpm.The time of culture of the present invention is preferably 2d-4d, more preferably 3d.
The present invention also provides the degrading tobacco stalk microbial inoculums to prepare the application in tabacco straw compost.
The preparation method of tabacco straw compost of the present invention, preferably includes: by the microbial inoculum and tabacco straw according to 5:
100 mass ratio is uniformly mixed, and the water content for adjusting mixture is 50~60%, starts compost.The present invention is in the compost
In the process, preferred gravity-flow ventilation, and daily periodic monitor heap temperature, temperature measuring point are located in the middle part of heap body.To heap temperature
When rising to 70 DEG C or so, into the preceding decomposed stage, preferred 4~5d turning is primary；To heap temperature be down to 50 DEG C and it is following when,
It is primary every 2d turning, until stopping turning when heap temperature is down to 40 DEG C or less and is no longer risen.The decomposed rank after the entrance of heap body
Section, the mass ratio of carbon and ammonia element in daily periodic monitor heap body, when the mass ratio of carbon and protium is less than 20,
Then show that the rear decomposed stage completes, entire composting process also terminates therewith.Bacillus amyloliquefaciens SL-7 of the present invention is enough fast
Fast degraded cellulose, and the ability with this strain with phosphorus decomposing, can effectively improve the phosphorus content in soil.
Bacillus amyloliquefaciens SL-7 provided by the invention and its application are described in detail below with reference to embodiment,
But they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Tabacco straw is acquired from the domestic tobacco leaf producing region in Anhui, 3~5cm long is shredded into after natural drying, with the omnipotent powder of high speed
Broken machine crushes, and after smashed tobacco rod powder crosses 50 meshes, is saved in spare in valve bag.
1g tobacco rod sample is weighed into the test tube of sterilizing, then plus 10mL sterilizing deionized water, be placed in isothermal vibration training
220rpm concussion 6~8h of leaching in case is supported, takes leaching liquid gradient dilution to 10 after the completion of concussion-1, 10-2。
The tobacco rod sample leaching liquor after 200 μ L dilution is drawn with liquid-transfering gun, respectively in bacterium primary dcreening operation culture medium (tabacco straw
Powder 10g/L, agar 18g/L, natural ph, 121 DEG C of sterilizing 20min) on be coated culture, bacterium primary dcreening operation culture medium is placed
It is cultivated in 37 DEG C of constant incubators.
The bacterium bacterial strain for the different shape that picking is grown on primary dcreening operation culture medium is to LB culture medium (tryptone 10g/L, ferment
Female bacterium cream 5g/L, agar 18g/L, NaCl 10g/L, natural ph, 121 DEG C of sterilizing 20min) on carry out separation scribing line.It will separation
Obtained single colonie of crossing is transferred to LB slant medium (tryptone 10g/L, saccharomycete cream 5g/L, agar 18g/L, NaCl
10g/L, natural ph are dispensed into slant tube mesohigh steam sterilizing (121 DEG C, 20min) after heating for dissolving, after the completion of sterilizing
Appropriate cooling is put into inclined-plane) on, it is stored in 4 DEG C of refrigerators in 37 DEG C of 3~5d of culture.
The bacterium that another picking is isolated, 37 DEG C, 220rpm is in LB liquid medium (tryptone 10g/L, saccharomycete cream
5g/L, NaCl 10g/L, natural ph, 121 DEG C of sterilizing 20min) in a certain amount of sterilizing is added after 8~12h of isothermal vibration culture
Glycerol be stored in -80 DEG C of refrigerators, final glycerol concentration should be between 20~25%.
Embodiment 2
Cellulose degradation aptitude tests are carried out to a variety of bacterium that embodiment 1 filters out:
1, culture medium is used in test
(1) bacterial strain purifies common culture medium
LB culture medium, LB liquid medium；
(2) degraded cellulose enzyme screening and culturing medium
Sodium carboxymethylcellulose culture medium (g/L): MgSO4·7H2O 0.1, KH2PO40.5, CaCl20.1, K2HPO42,
CMC-Na 10, (NH4)2SO42, agar 18, pH 7.0~7.4；
Carboxymethylcelluloenzyme enzyme (CMCase) living detection culture medium (g/L): peptone 10, beef extract 10, sodium chloride 1.5,
CMC-Na 10, KH2PO41.0, MgSO4·7H2O 0.3, pH 7.0；
Filter paper enzyme activity (FPase) detects culture medium (g/L): peptone 10, beef extract 10, sodium chloride 1.5, filter paper 0.05,
KH2PO41.0, MgSO4·7H2O 0.3, pH 7.0.
2, main agents
Congo red solution (1mg/mL): weighing that 1g is Congo red to be settled to 1000mL, and 4 DEG C of refrigerators save backup.
Sodium chloride solution (1mol/L): it weighs 58.5g sodium chloride and is settled to 1000mL, 4 DEG C of refrigerators save backup.
Buffer solution: with taking 50mL buffer solution to measure respectively with 0.2mol/L sodium acetate solution and 0.2mol/L acetum
Take 35.2mL and 14.8mL.
DNS reagent: weighing 3,5-dinitrosalicylic acid of 6.3g, and 21.0g NaOH is in 500mL beaker, with a small amount of distillation
Water dissolution and 500mL contain the hydrothermal solution of 185g sodium potassium tartrate tetrahydrate, add 5g phenol and 5g anhydrous sodium sulfite, most
After be settled to 1000mL, be fitted into brown reagent bottle, save a week stablize after it is spare.
Sodium carboxymethylcellulose substrate solution (0.5%): 5g sodium carboxymethylcellulose is dissolved in 1000mL buffer solution, standby
With.
Filter paper substrate solution: 0.03g filter paper is added in 2mL buffer solution.
3, test method
(1) plate qualitative determination
By single colonie difference dibbling on sodium carboxymethylcellulose culture medium, 27 DEG C of culture 3d of bacterium.With the 1mg/mL the Congo
Red colouring 15min, incline dye liquor, adds the sodium chloride solution rinsing of lmol/L, outwells sodium chloride solution after 15min, survey saturating
The diameter and colony diameter of bright circle: this plant of bacterium can grow on CMC-Na culture medium, generate after congo red staining apparent
Cellulose degradation circle, as a result as shown in Figure 1, the biggish bacterium of ratio of degradation loop diameter and colony diameter is Xie Dian after culture 4d
Afnyloliquefaciens SL-7 (7.52 ± 0.35).Measurement cellulase activity is gone with the bacillus amyloliquefaciens SL-7 again.
(2) measurement of cellulase activity
DNS method measures glucose light absorption value, with OD530It is that ordinate draws grape for abscissa, glucose amount (mg/mL)
Standard for Sugars curve obtains regression equation y=0.452x+0.016, R2=0.9916.By the higher strain inoculated CMCase of bacteriostasis rate
It detects culture medium and FPase detects culture medium, distinguish shaken cultivation 1,2,3,4,5,6,7d, 5000r/ in 28 DEG C, 160r/min
Min is centrifuged 10min, and supernatant is taken to detect CMC enzyme activity and FPase enzyme activity respectively.The definition of enzyme activity unit: it is fermented with every milliliter
The amount that supernatant enzymatic reaction 30min discharges 1 μ g glucose is an enzyme-activity unit (U).
CMCase measurement: 1mL 1%CMC-Na solution is added (with the second of 0.2mol/LpH 4.8 in 1mL fermented supernatant fluid
Acid buffer is prepared) in, the fermented supernatant fluid with boiling water inactivation 10min is control, 50 DEG C of enzymatic reactions after mixing well
30min is rapidly added DNS reagent 3mL after taking-up, be placed in boiling water bath colour developing 10min, flowing water is cooled to room temperature, then uses deionization
Water is settled to 25mL, shakes up, and is returned to zero with control tube, measures 530nm light absorption value, calculates reduced sugar according to glucose standard curve
Amount.
FPase measurement: 1mL fermented supernatant fluid is added to the Acetic acid-sodium acetate buffer of 1mL 0.2mol/L pH 4.8
In, and Xinhua's filter paper item (6cm × 1cm) is added, filter paper item, which is soaked in liquid, to be mixed, 50 DEG C of enzymatic reaction 30min, with boiling water
The fermented supernatant fluid for inactivating 10min is control, and DNS method measures reduced sugar production quantity.
As a result as shown in Fig. 2, this plant of bacterium is in CMCase and FPase culture medium culture, bacterial strain SL-7 fermented liquid supernatant
As time increases in the trend fallen after rising, CMCase reaches up to the CMCase and FPase of liquid in 3d
84.81U/mL, FPase reach up to 35.40U/mL in 3d.
Embodiment 3
Obtained bacterial strain SL-7 is screened to embodiment 2 carries out dissolving P capacity test:
1, culture medium
(1) bacterial strain purifies common culture medium
LB culture medium, LB liquid medium；
(2) phosphorus decomposing effect measuring culture medium
Organic phosphorus Phos solid medium (g/L): glucose 10g, (NH4)2SO40.5, NaCl0.3, KCl 0.3,
K2SO40.3,MgSO4·7H2O 0.3, FeSO4·7H2O 0.03, MnSO4·4H2O0.03, calcium phosphate 5.0, lecithin 2.0, fine jade
Rouge 20.0, pH are 7.0~7.5.
Organic phosphorus fluid nutrient medium (g/L): glucose 10g, (NH4)2SO40.5, NaCl 0.3, KCl 0.3, MgSO4·
7H2O 0.3, FeSO4·7H2O 0.03, MnSO4·4H2O 0.03, CaCO35.0, lecithin 2.0, pH is 7.0~7.5.It is (real
Lecithin is replaced with fresh egg yolk liquid when testing, fresh egg yolk liquid 3mL, egg yolk liquid and 0.9% physiological saline etc. is added in every 50mL
Ratio is prepared.)
Phos fluid nutrient medium (g/L): glucose 10.0, (NH4)2SO40.5, NaCl 0.3, KCl 0.3, MgSO4·
7H2O 0.3, FeSO4·7H2O 0.03, MnSO4·4H2O 0.03, Ca3(PO4)25, pH be 7.0~7.5.
2, main agents
(1) reagent needed for phosphorus decomposing effect measuring
Antimony tartrate potassium solution (0.5%): weighing 0.5g potassium antimony tartrate solid and be added in 100mL distilled water, stirring,
Mixing.
The anti-storage liquid of molybdenum antimony: concentrated sulfuric acid 153mL is slowly poured into the beaker for being equipped with about 400mL distilled water, stirring, cold
But.10g ammonium molybdate is dissolved in about 60 DEG C of 300mL distilled water, cooling.Then concentrated sulfuric acid solution is poured into ammonium molybdate solution
In, add 100mL0.5% potassium antimony tartrate (KSB, C4H4O6·1/2H2O) solution is finally settled to 1L with distilled water, keeps away
Light saves.
The anti-color developing agent of molybdenum antimony: 1.5g ascorbic acid (C6H8O6, left-handed) and it is dissolved in the anti-storage liquid of 100mL molybdenum antimony.
3, SL-7 dissolving P capacity measures
(1) plate qualitative determination
By single colonie difference dibbling on phosphate solubilizing bacteria isolation medium, after 37 DEG C of 3~5d of culture, as a result as shown in figure 3, seeing
The case where transparent circle generates is examined, the transparent loop diameter of phosphorus decomposing (D) is measured and recorded.
(2) content of titanium pigment is quantitative determined
Using molybdenum antimony resistance colorimetric method, water-soluble phosphorus content in SL-7 culture solution is measured.With visible spectrophotometer in wave
Light absorption value is measured at long 720nm, with the content of quantitative analysis titanium pigment.
Phosphorus Specification Curve of Increasing: being separately added into the standard phosphorus solution of respective volume 100mg/L in volumetric flask, adds 2 drops 2,6
One dinitrophenol makees indicator, adjusts pH value with the NaOH solution of dilute sulfuric acid and 10%, adds the anti-color developing agent 5mL of aluminium antimony, constant volume
To scale, making standard phosphorus concentration is respectively 0,0.2,0.4,0.6,0.8 and 1.0mg/L, is shaken up, and (25 DEG C or so) are anti-at room temperature
Ultraviolet specrophotometer colorimetric at 720nm is used after answering 30min, standard curve is drawn according to result.
The measurement of solvable phosphorus content in culture solution: SL-7 is inoculated into organic phosphorus (or Phos) fluid nutrient medium of 50mL
In, it is compared with not connecing bacterium, shaking speed 150r/min, 28 DEG C of culture 3d.Culture solution is transferred to sterile 50mL centrifuge tube
In, ultrasonic cell-break is carried out using numerical control ultrasonic cleaner, 20min is crushed, is allowed to release intracellular available phosphorus,
It is centrifuged after 20min with the revolving speed of 4000r/min and takes 2.5mL supernatant in 50mL volumetric flask plus 2 drop 2,6- dinitrophenol works
Indicator, add a drop dilute sulfuric acid to reaction solution be it is colourless, add the anti-color developing agent 5mL of molybdenum antimony, constant volume, reaction.Use uv-spectrophotometric
OD value of the meter measurement supernatant at 720nm.The available phosphorus content in supernatant is obtained according to standard curve.As a result such as 1 institute of table
Show:
1 dissolving P capacity test result of table
By Fig. 3 and table 1 it is found that bacterial strain SL-7 shows apparent phosphorus decomposing on Phos and organic phosphorus culture medium flat plate
Circle, to tentatively judge that bacterial strain SL-7 has certain dissolving P capacity.
Embodiment 4
For influence of the measurement oxygen content to bacterial strain SL-7 producing enzyme, be set separately shaking speed be 140rpm, 160rpm,
180rpm, 200rpm and 220rpm, according to 4% inoculum concentration (accessing 4mL bacteria suspension in every 100mL CMC enzymatic production culture medium)
It is inoculated in 100mL enzymatic production culture medium.The shaking table shaken cultivation under 37 DEG C of parts, and CMCase, FPase are measured on day 3.
As a result as shown in Figure 4: with the increase of fermentation liquid revolving speed, the presentation of strain enzyme-producing activity first rises the variation to tend to balance afterwards and becomes
Gesture, FPase and CMCase highest when fermentation liquid revolving speed is 200 are 40.93U/mL and 106.56U/mL, to sum up, the bacterium it is most suitable
Producing enzyme revolving speed is 200rpm, and the best producing enzyme range of speeds is 180~200rpm.
Embodiment 5
Influence for measuring temperature to bacterial strain SL-7 producing enzyme, it is 28 DEG C, 37 DEG C, 46 DEG C, 55 DEG C that shaking table temperature, which is set separately,
With 60 DEG C, according to 4% inoculum concentration (in every 100mL CMC enzymatic production culture medium access 4mL bacteria suspension), be inoculated in 100mL hair
In ferment culture medium.Shaking table shaken cultivation under the conditions of revolving speed is 200rpm, and CMCase, FPase are measured on day 3.Knot
Fruit is as shown in Figure 5: as broth temperature increases, the presentation of strain enzyme-producing activity first rises the change for declining afterwards and finally tending to balance
Change trend.FPase and CMCase highest when broth temperature is 37 DEG C are 39.09U/mL and 108.86U/mL；To sum up, the bacterium
Most suitable producing enzyme temperature be 37 DEG C, best producing enzyme temperature range be 25~45 DEG C.
Embodiment 6
It is 2%, 4%, 6%, 8%, 10% and 12% (in every 100mLCMC enzymatic production culture medium that inoculum concentration, which is set separately,
It is respectively connected to 2mL, 4mL, 6mL, 8mL, 10mL, 12mL bacteria suspension) it is inoculated in 100mLCMC enzymatic production culture medium.37
DEG C, shaking table shaken cultivation under the conditions of 200rpm, and measure CMCase, FPase respectively on day 3.As a result as shown in Figure 6: with
It is inoculated with the increase of volume, the presentation of strain enzyme-producing activity first rises the variation tendency for declining afterwards and finally tending to balance, works as inoculum
When product is 8%, there is peak for 40.94U/mL and 115.96U/mL in FPase and CMCase；To sum up, the most suitable producing enzyme of the bacterium
Being inoculated with volume is 8%, and best producing enzyme inoculation volume range is 6~10%.
Embodiment 7
Bacteria suspension is inoculated in 100mL enzymatic production culture medium by 8% inoculum concentration, it is initial to adjust enzymatic production culture medium
PH is respectively 5.0,6.0,7.0,8.0 and 9.0.Shaking table shaken cultivation under the conditions of 37 DEG C, 200rpm, and on day 3 and measurement
CMCase,FPase.As a result as shown in Figure 7: as fermentation liquid PH increases, the presentation of strain enzyme-producing activity first rises to be declined and most afterwards
The variation tendency to tend to balance eventually.CMCase and FPase highest when fermentation liquid PH is 7 are 114.31U/mL and 54.57U/mL；
To sum up, the most suitable producing enzyme PH of the bacterium is 7, and best producing enzyme PH range is 6~8.
Embodiment 8
Influence for measurement different nitrogen sources to strain enzyme-producing, it is 0.4% peptone, 0.4% ammonium sulfate that nitrogen source, which is set separately,
With 0.4% urea, and bacteria suspension is inoculated in 100mLCMC enzymatic production culture medium by 8% inoculum concentration.At 37 DEG C, 200rpm
Under the conditions of shaking table shaken cultivation, and on day 3 respectively measure CMCase, FPase.As a result as shown in Figure 8: in peptone processing,
CMCase and FPase can respectively reach 119.1U/mL, 75.22U/mL: in Urea treatment, CMCase and FPase can be respectively reached
59.73U/mL, 9.22U/mL: in ammonium sulfate processing, CMCase and FPase can respectively reach 75.96/mL, 15.87U/mL.Egg
CMCase and FPase in white peptone processing will be high compared to urea and ammonium sulfate, it can be seen that peptone can promote bacterial strain production
Enzyme, and urea and ammonium sulfate produce different degrees of inhibiting effect to strain enzyme-producing.
Embodiment 9
Influence for measurement different carbon source to strain enzyme-producing, it is 0.5% glucose, 0.5%CMC-Na that carbon source, which is set separately,
With 1% tabacco straw powder (0.5% soluble carbon source or l% insolubility carbon source), and by bacteria suspension by the inoculation of 8% inoculum concentration
In 100mL CMC enzymatic production culture medium.At 37 DEG C, shaking table shaken cultivation under the conditions of 200rpm, and measure respectively on day 3
CMCase,FPase.As a result as shown in Figure 9: in CMC-Na processing, CMCase and FPase can respectively reach 123.89U/mL,
90.71U/mL；.CMCase and FPase can respectively reach 105.46U/mL, 53.84U/mL in tabacco straw powder-processed；Grape
In sugar processing, CMCase and FPase can respectively reach 59.73U/mL, 38.35U/mL.CMC-Na processing in CMCase and
FPase will be high compared to tabacco straw powder and glucose, it can be seen that CMC-Na can promote strain enzyme-producing and tabacco straw
Powder and glucose generate different degrees of inhibiting effect to strain enzyme-producing.
Embodiment 10
To measure the influence of different carbon-nitrogen ratios to strain enzyme-producing, the most suitable carbon source of optimization and most suitable is chosen in above-mentioned experiment
Nitrogen source, it is 2:1,1:1 and 1:2 that carbon-nitrogen ratio, which is set separately, and bacteria suspension is inoculated in the training of 100mL enzymatic production by 8% inoculum concentration
It supports in base.Shaking table shaken cultivation under the conditions of 37 DEG C, 200rpm, and CMCase, FPase are measured on day 3.As a result such as Figure 10 institute
Showing that: C/N is in 2:1 processing, CMCase and FPase can respectively reach 135.69U/mL, 67.85U/mL:C/N and be in 1:1 processing,
It is in 1:2 processing that CMCase and FPase, which can respectively reach 120.21U/mL, 87.02U/mL:C/N, and CMCase and FPase can divide
84.07U/mL, 103.98U/mL are not reached.C/N is that the CMCase in 2:1 processing is 1:1 compared to C/N and C/N is that 1:2 will
It is high, it can be seen that C/N is that 2:1 can promote strain enzyme-producing, and C/N is 1:1 and C/N is that 1:2 produces difference to strain enzyme-producing
The inhibiting effect of degree.
Embodiment 11
1, the building of tabacco straw high-effect bacterial
SL-7 is inoculated in peptone as only nitrogen source, CMC-Na in culture substrate as sole carbon in culture substrate
Source and C/N are in 2:1CMC fermentation medium, in most suitable producing enzyme culture environment condition are as follows: pH7.0, inoculation volume 8%, revolving speed
200rpm, 37 DEG C of temperature cultivate three days, construct tabacco straw high efficiency degradation bacterial agent.
2, composting tabacco straw compost
The addition of 2.1 microbial inoculums
Microbial inoculum is added in watering can.Then tabacco straw composting material is spread out, then with watering can microbial inoculum according to cigarette
The quality proportioning of careless straw compost raw material 5:100 is uniformly sprayed at the tabacco straw composting material surface spread out.After sprayed,
It is stirred for uniformly, and adjusting mixing water content of matter is 50% -60%, starts compost.
2.2 heap body compostings
The composting material for having added microbial inoculum is filled into simple composting device, high about 0.56m, volume about 60L is made in heap
Heap body.During composting.Daily periodic monitor heap temperature, temperature measuring point are located in the middle part of heap body.Gravity-flow ventilation, to heap body temperature
When degree rises to 70 DEG C or so, into the preceding decomposed stage, turning in 4-5 days is primary.50 DEG C are down to heap temperature and hereinafter, every 2
Its turning is primary, until stopping turning when heap temperature is down to 40 DEG C or less and is no longer risen.The decomposed stage after the entrance of heap body,
The mass ratio of carbon and nitrogen element in daily periodic monitor heap body, when carbon and the mass ratio of nitrogen are less than 20
When, then show that the rear decomposed stage completes, entire composting process also terminates therewith.
The present invention provides a bacillus amyloliquefaciens SL-7, there is bacterial strain SL-7 powerful degraded cellulose to conciliate
Phosphorus ability can be used for preparing tabacco straw compost.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Fuyang Teachers College
<120>one bacillus amyloliquefaciens SL-7 and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1429
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 1
tataatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt 60
gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat 120
accggatggt tgtntgaacc gcatggttca gacataaaag gtggcttcgg ctaccactta 180
cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg 240
cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 360
tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc 420
aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480
gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 540
ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600
tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 660
agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 720
cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780
tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc 840
ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct 960
gacaatccta gagataggac gtccccttcg ggggcagagt gacaggtggt gcatggttgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt 1080
agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt 1140
ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200
cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc 1260
ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca 1320
tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380
taacacccga agtcggtgag gtaaccttta tggagccagc cgccgaagg 1429
Claims (10)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SL-7, deposit number is GDMCC No:
60630。
2. bacillus amyloliquefaciens SL-7 according to claim 1, which is characterized in that the bacillus amyloliquefaciens SL-7's
16S gene order is as shown in SEQ ID NO.1.
3. application of the bacillus amyloliquefaciens SL-7 as claimed in claim 1 or 2 in degraded cellulose.
4. application of the bacillus amyloliquefaciens SL-7 as claimed in claim 1 or 2 in phosphorus decomposing.
5. bacillus amyloliquefaciens SL-7 as claimed in claim 1 or 2 is preparing the application in degrading tobacco stalk microbial inoculum.
6. applying according to claim 5, which is characterized in that the preparation method of the degrading tobacco stalk microbial inoculum, including with
Lower step: the bacillus amyloliquefaciens SL-7 being inoculated in CMC fermentation medium and cultivates 3d, obtains degrading tobacco stalk bacterium
Agent；It using peptone is only nitrogen source, CMC-Na for sole carbon source in the CMC fermentation medium, the sole carbon source and unique
The C/N ratio of nitrogen source is 2:1.
7. applying according to claim 6, which is characterized in that the inoculation volume of the bacillus amyloliquefaciens SL-7 is described
The 6~10% of CMC fermentation medium volume.
8. applying according to claim 6, which is characterized in that the temperature of the culture be 25~45 DEG C, pH value be 6.0~
8.0。
9. applying according to claim 8, which is characterized in that with stirring, the revolving speed of the stirring in the incubation
For 180~200rpm.
10. any one of the claim 5~9 degrading tobacco stalk microbial inoculum is preparing the application in tabacco straw compost.
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| CN111500487A (en) * | 2020-03-20 | 2020-08-07 | 阜阳师范大学 | Compound microorganism, microbial inoculum and preparation method and application thereof |
| CN114134077A (en) * | 2021-11-19 | 2022-03-04 | 江苏科技大学 | Silkworm excrement-derived cellulose degrading bacterium DC11 and screening method and application thereof |
| CN114480169A (en) * | 2021-12-24 | 2022-05-13 | 鞍钢集团矿业有限公司 | Bacillus amyloliquefaciens and application thereof |
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| CN111073833A (en) * | 2019-12-27 | 2020-04-28 | 北京工商大学 | Bacillus amyloliquefaciens, microbial inoculum, application thereof and method for degrading formaldehyde |
| CN111500487A (en) * | 2020-03-20 | 2020-08-07 | 阜阳师范大学 | Compound microorganism, microbial inoculum and preparation method and application thereof |
| CN114134077A (en) * | 2021-11-19 | 2022-03-04 | 江苏科技大学 | Silkworm excrement-derived cellulose degrading bacterium DC11 and screening method and application thereof |
| CN114480169A (en) * | 2021-12-24 | 2022-05-13 | 鞍钢集团矿业有限公司 | Bacillus amyloliquefaciens and application thereof |
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