CN110079481B

CN110079481B - Bacillus amyloliquefaciens S L-7 and application thereof

Info

Publication number: CN110079481B
Application number: CN201910423005.8A
Authority: CN
Inventors: 盛良全; 梅金飞; 吴福芳; 徐华杰; 李文胜; 刚利萍; 余梅霞; 戴亚
Original assignee: Fuyang Normal University
Current assignee: Fuyang Normal University
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2020-08-04
Anticipated expiration: 2039-05-21
Also published as: CN110079481A

Abstract

The invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) S L-7 and application thereof, and relates to the technical field of microorganisms, wherein the Bacillus amyloliquefaciens S L-7 is screened from tobacco straw waste and has a preservation number of GDMCC No: 60630. in the embodiment of the invention, the Bacillus amyloliquefaciens S L-7 can generate obvious cellulose degradation rings, CMCase can reach 84.81U/m L and 35.40U/m L in a cellulase activity test, and organic phosphorus and inorganic phosphorus show obvious phosphorus degradation rings in a phosphorus degradation test, so the Bacillus amyloliquefaciens S L-7 can be used for degrading cellulose and improving the phosphorus content in soil, and the tobacco straw compost is prepared.

Description

Bacillus amyloliquefaciens S L-7 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus amyloliquefaciens S L-7 and application thereof.

Background

China is the country with the largest tobacco cultivation area in the world, the yield of tobacco leaves is 450-500 ten thousand tons every year, and meanwhile, a large amount of waste tobacco stems are generated. The content of organic matters in the tobacco straws is about 90-95%, the content of mineral matters is 5-10%, wherein the content of nitrogen is 0.5-1.2%, the content of phosphorus is 0.1-0.25%, and the content of potassium is 0.8-1.8%; meanwhile, the straws also contain various microorganisms, but the microorganisms screened from the tobacco straws at present mostly have single functions, or degrade cellulose, or degrade nicotine, or are used for producing laccase, and no microorganism which is screened from the tobacco straws and has multiple functions is provided.

Disclosure of Invention

In view of the above, the invention aims to provide a bacillus amyloliquefaciens S L-7 and application thereof, wherein the bacillus amyloliquefaciens S L-7 is screened from tobacco straw wastes, and has the functions of degrading cellulose and dissolving phosphorus.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) S L-7 with a collection number of GDMCC No. 60630.

Preferably, the 16S gene sequence of the Bacillus amyloliquefaciens S L-7 is shown as SEQ ID NO. 1.

The invention also provides application of the bacillus amyloliquefaciens S L-7 in cellulose degradation.

The invention also provides application of the bacillus amyloliquefaciens S L-7 in phosphate solubilizing.

The invention also provides application of the bacillus amyloliquefaciens S L-7 in preparation of a tobacco straw degrading microbial inoculum.

Preferably, the preparation method of the tobacco straw degrading microbial inoculum comprises the following steps of inoculating the bacillus amyloliquefaciens S L-7 into a CMC fermentation culture medium to be cultured for 3d to obtain the tobacco straw degrading microbial inoculum, wherein peptone is used as a unique nitrogen source, CMC-Na is used as a unique carbon source in the CMC fermentation culture medium, and the C/N ratio of the unique carbon source to the unique nitrogen source is 2: 1.

Preferably, the inoculation volume of the bacillus amyloliquefaciens S L-7 is 6-10% of the volume of the CMC fermentation medium.

Preferably, the temperature of the culture is 37 ℃, and the pH value is 6.0-8.0.

Preferably, stirring is carried out in the culture process, and the rotation speed of the stirring is 180-200 rpm.

The invention also provides application of the tobacco straw degrading microbial inoculum in preparation of tobacco straw compost.

The invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) S L-7, wherein the Bacillus amyloliquefaciens S L-7 is screened from tobacco straw wastes, in the embodiment of the invention, experiments prove that the Bacillus amyloliquefaciens S L-7 can generate an obvious cellulose degradation circle, in a cellulase activity test, the CMCase can reach 424.04U/m L and can reach 176.99U/m L, in a phosphate solubilizing test, organic phosphorus and inorganic phosphorus can both show an obvious phosphate solubilizing circle, and the Bacillus amyloliquefaciens S L-7 can be used for degrading cellulose and solubilizing phosphorus so as to prepare tobacco straw compost.

Biological preservation information

The Bacillus amyloliquefaciens strain is characterized in that the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens S L-7, the preservation place is Guangdong province microorganism strain preservation center, the preservation address is No. 59 building 5 of Miao No. 100 of Fujiu, Mieli, Guangzhou city, the preservation time is 4-11 days in 2019, and the preservation number is GDMCC No. 60630.

Drawings

FIG. 1 is a graph showing the hydrolysis effect of the S L-7 strain on Congo red medium;

FIG. 2 is a graph showing the results of cellulase activity measurement;

FIG. 3 is a graph showing the results of measurement of phosphorus-solubilizing ability;

FIG. 4 shows the effect of rotational speed on the total enzyme activity of S L-7 strain for enzyme production;

FIG. 5 shows the effect of culture temperature on the total enzyme activity of S L-7 strain for enzyme production;

FIG. 6 shows the effect of inoculum volume on total enzyme activity for enzyme production of S L-7 strain;

FIG. 7 is a graph showing the effect of initial pH on the total enzyme activity of enzyme production by S L-7 strain;

FIG. 8 shows the effect of different carbon sources on the total enzyme activity of S L-7 strain;

FIG. 9 shows the effect of different nitrogen sources on the total enzyme activity of S L-7 strain;

FIG. 10 shows the effect of different carbon-nitrogen ratios on the total enzyme activity for producing enzyme from S L-7 strain.

Detailed Description

The invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) S L-7 with a preservation number of GDMCC No. 60630. the 16S gene sequence of the Bacillus amyloliquefaciens S L-7 is shown in SEQ ID NO. 1.

The preparation method of the tobacco straw degrading microbial inoculum preferably comprises the following steps of inoculating bacillus amyloliquefaciens S L-7 into a CMC fermentation culture medium to be cultured for 3d to obtain the tobacco straw degrading microbial inoculum, wherein peptone is used as a unique nitrogen source, CMC-Na is used as a unique carbon source in the CMC fermentation culture medium, the C/N ratio of the unique carbon source to the unique nitrogen source is 2:1, the inoculation volume of the bacillus amyloliquefaciens S L-7 is preferably 6-10% of the volume of the CMC fermentation culture medium, more preferably 8%, the culture temperature is preferably 25-45 ℃, more preferably 37 ℃, the culture pH value is preferably 6.0-8.0, more preferably 7.0, the culture process of the invention is preferably accompanied with stirring, the stirring rotation speed is preferably 180-200 rpm, more preferably 200rpm, the culture time is preferably 2d-4d, and more preferably 3 d.

The preparation method of the tobacco straw compost preferably comprises the steps of uniformly mixing the microbial inoculum and tobacco straws according to the mass ratio of 5:100, adjusting the water content of the mixture to be 50-60%, and starting composting, wherein in the composting process, natural ventilation is preferably adopted, the temperature of a compost is regularly monitored every day, a temperature measuring point is positioned in the middle of the compost, when the temperature of the compost is increased to about 70 ℃, the compost enters a pre-composting stage, the compost is turned for 4-5 d preferably, when the temperature of the compost is reduced to 50 ℃ or below, the compost is turned for 2d every time, when the temperature of the compost is reduced to 40 ℃ or below, the turning is stopped, when the temperature of the compost enters a post-composting stage, the mass ratio of carbon elements and ammonia elements in the compost is regularly monitored every day, the post-composting stage is finished, the whole composting process is finished, and the bacillus amyloliquefaciens S L-7 can quickly degrade cellulose, has the capacity of decomposing phosphorus, and can effectively improve the phosphorus content in soil.

The present invention provides Bacillus amyloliquefaciens S L-7 and its applications as described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Collecting tobacco straws from a tobacco leaf producing area in Anhui province, naturally drying the tobacco straws, cutting the tobacco straws into 3-5 cm long tobacco straws, crushing the tobacco straws by using a high-speed universal crusher, and storing the crushed tobacco straw powder into a self-sealing bag for later use after the crushed tobacco straw powder passes through a 50-mesh sieve.

Weighing 1g of tobacco stem sample into a sterilized test tube, adding 10m L sterilized deionized water, placing the test tube into a constant-temperature shaking incubator at 220rpm, shaking and leaching for 6-8 h, and after shaking is finished, taking leaching solution and diluting the leaching solution to 10 degrees centigrade in gradient^-1，10^-2。

Sucking 200 mu L diluted tobacco stem sample leaching solution with a pipette, respectively coating and culturing on bacterial primary screening culture medium (tobacco stem powder 10 g/L, agar 18 g/L, natural pH, sterilizing at 121 deg.C for 20min), and culturing in 37 deg.C constant temperature incubator.

Selecting bacterial strains with different forms growing on a primary screening culture medium, carrying out separation and streaking on L B culture medium (tryptone 10 g/L, yeast extract 5 g/L, agar 18 g/L10 g/L, natural pH value, sterilization at 121 ℃ for 20min), transferring a single colony obtained by separation and streaking to L B slant culture medium (tryptone 10 g/L, yeast extract 5 g/L, agar 18 g/L10 g/L, natural pH value, heating and dissolving, subpackaging the mixture into a test tube, carrying out high-pressure steam sterilization (121 ℃, 20min), cooling and placing the mixture into a slant after sterilization, and culturing at 37 ℃ for 3-5 d and storing in a 4 ℃ refrigerator.

And (2) additionally selecting separated bacteria, carrying out constant-temperature shaking culture in L B liquid culture medium (tryptone 10 g/L, yeast extract 5 g/L10 g/L, natural pH value and sterilization at 121 ℃ for 20min) at 37 ℃ and 220rpm for 8-12 h, adding a certain amount of sterilized glycerol, and storing in a refrigerator at-80 ℃ until the final concentration of the glycerol is 20-25%.

Example 2

The cellulose-degrading ability of the various bacteria selected in example 1 was tested:

1. culture medium for test

(1) Common culture medium for strain purification

L B medium, L B liquid medium;

(2) screening culture medium for degrading cellulase

Sodium carboxymethylcellulose medium (g/L): MgSO₄·7H₂O 0.1，KH₂PO₄0.5，CaCl₂0.1，K₂HPO₄2，CMC-Na 10，(NH₄)₂

SO

₄2, agar 18, and pH 7.0-7.4;

carboxymethyl cellulose enzyme activity (CMCase) detection medium (g/L) comprises peptone 10, beef extract 10, sodium chloride 1.5, CMC-Na10, KH₂PO₄1.0，MgSO₄·7H₂O 0.3，pH 7.0；

Filter paper enzyme activity (FPase) detection 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. Primary reagent

Congo red solution (1mg/m L) weighing 1g of Congo red to 1000m L, and storing in refrigerator at 4 deg.C for use.

Sodium chloride solution (1 mol/L) weighing 58.5g sodium chloride to 1000m L, and storing in refrigerator at 4 deg.C for use.

The buffer solution is prepared by taking 50m L buffer solution and measuring 35.2m L and 14.8m L by 0.2 mol/L sodium acetate solution and 0.2 mol/L acetic acid solution respectively.

Weighing 6.3g of 3, 5-dinitrosalicylic acid and 21.0g of NaOH in a 500m L beaker, dissolving the 3, 5-dinitrosalicylic acid and 21.0g of NaOH in a small amount of distilled water, adding 5g of phenol and 5g of anhydrous sodium sulfite into 500m L hot water solution containing 185g of potassium sodium tartrate, finally fixing the volume to 1000m L, filling the mixture into a brown reagent bottle, and storing the mixture for later use after the mixture is stable for one week.

Sodium carboxymethylcellulose substrate solution (0.5%) 5g of sodium carboxymethylcellulose are dissolved in 1000m L buffer solution for use.

Filter paper substrate solution 0.03g of filter paper was added to 2m L buffer solution.

3. Test method

(1) Plate qualitative determination

Respectively dibbling a single colony on a sodium carboxymethylcellulose culture medium, culturing the bacteria at 27 ℃ for 3d, staining the bacteria for 15min by using 1mg/m L Congo red, pouring off a staining solution, adding a sodium chloride solution of lmol/L for rinsing, pouring off the sodium chloride solution after 15min, measuring the diameter of a transparent ring and the diameter of the colony, wherein the bacteria can grow on the CMC-Na culture medium and generate an obvious cellulose degradation ring after Congo red staining, and the result is shown in figure 1, the bacteria with the larger ratio of the diameter of the degradation ring to the diameter of the colony after 4d culture is Bacillus amyloliquefaciens S L-7 (7.52 +/-0.35), and then the activity of the cellulase is measured by using the Bacillus amyloliquefaciens S L-7.

(2) Determination of cellulase Activity

Determination of absorbance value of glucose by DNS method, as OD₅₃₀Drawing a glucose standard curve by using an abscissa and a glucose amount (mg/m L) as an ordinate, obtaining a regression equation y of 0.452x +0.016, and R2 of 0.9916, inoculating the strain with higher bacteriostatic rate into a CMCase detection culture medium and an FPase detection culture medium, respectively carrying out shaking culture at 28 ℃ and 160R/min for 1, 2, 3, 4, 5, 6 and 7d, centrifuging at 5000R/min for 10min, and respectively taking supernatant to detect the activity of CMC enzyme and the activity of FPase.

CMCase determination, namely adding 1m L fermentation supernatant into 1m L1% CMC-Na solution (prepared by 0.2 mol/L of acetic acid buffer solution with pH 4.8), taking the fermentation supernatant inactivated by boiling water for 10min as a control, carrying out enzymatic reaction at 50 ℃ for 30min after fully mixing, taking out, quickly adding DNS reagent 3m L, placing in a boiling water bath for color development for 10min, cooling running water to room temperature, fixing the volume to 25m L by deionized water, shaking up, adjusting zero by using a control tube, determining the light absorption value at 530nm, and calculating the amount of reducing sugar according to a glucose standard curve.

FPase determination comprises adding 1m L fermentation supernatant into 1m L0.2.2 mol/L pH 4.8 acetic acid-sodium acetate buffer solution, adding Xinhua filter paper strip (6cm × 1cm), soaking filter paper strip in the solution, mixing, performing enzymatic reaction at 50 deg.C for 30min, and determining reducing sugar production amount by DNS method with fermentation supernatant inactivated with boiling water for 10min as control.

As shown in FIG. 2, the strain shows that the CMCase and the FPase of the strain S L-7 fermentation broth supernatant both have a tendency of rising first and falling second with the time increase in the CMCase and FPase culture medium, and the CMCase reaches the highest 84.81U/m L at the 3d and reaches the highest 35.40U/m L at the 3 d.

Example 3

The strain S L-7 screened in example 2 was tested for its phosphate solubilizing ability:

1. culture medium

(1) Common culture medium for strain purification

L B medium, L B liquid medium;

(2) phosphate solubilizing effect determination culture medium

Organic phosphorus inorganic phosphorus solid culture medium (g/L), glucose 10g, (NH)₄)₂SO₄0.5，NaCl0.3，KCl 0.3，K₂SO₄0.3,MgSO₄·7H₂O 0.3，FeSO₄·7H₂O 0.03，MnSO₄·4H₂0.03 percent of O, 5.0 percent of calcium phosphate, 2.0 percent of lecithin and 20.0 percent of agar, and the pH value is 7.0-7.5.

Organophosphorus liquid Medium (g/L), glucose 10g, (NH)₄)₂SO₄0.5，NaCl 0.3，KCl 0.3，MgSO₄·7H₂O 0.3，FeSO₄·7H₂O 0.03，MnSO₄·4H₂O 0.03，CaCO₃5.0, 2.0 lecithin and 7.0-7.5 (in the experiment, fresh egg yolk liquid is used for replacing lecithin, 3m L of fresh egg yolk liquid is added into every 50m L, and the egg yolk liquid and 0.9% of physiological saline are prepared in equal proportion.)

Inorganic phosphorus liquid cultureBase (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, the pH value is 7.0-7.5.

2. Primary reagent

(1) Reagent for determining phosphate solubilizing effect

0.5g of antimony potassium tartrate solution (0.5 percent) is weighed and added into 100m L distilled water, stirred and mixed evenly.

The molybdenum-antimony anti-storage solution is prepared by slowly pouring 153m L g of concentrated sulfuric acid into a beaker containing 400m L g of distilled water, stirring, cooling, dissolving 10g of ammonium molybdate in 300m L of distilled water at 60 ℃, cooling, pouring concentrated sulfuric acid solution into ammonium molybdate solution, and adding 100m L0.5.5% of potassium antimonium tartrate (KSB, C)₄H₄O₆·1/2H₂O) solution, finally adding distilled water to reach the volume of 1L, and storing in dark place.

Molybdenum antimony color-developing resisting agent: 1.5g ascorbic acid (C)₆H₈O₆Levorotatory) was dissolved in 100m L Mo-Sb stock solution.

3. Determination of phosphate solubilizing ability of S L-7

(1) Plate qualitative determination

And respectively dibbling the single colony on a phosphate solubilizing bacteria separation culture medium, culturing at 37 ℃ for 3-5D, observing the generation condition of the transparent ring as shown in figure 3, and measuring and recording the diameter (D) of the phosphate solubilizing transparent ring.

(2) Quantitative determination of soluble phosphorus content

And measuring the content of water-soluble phosphorus in the culture solution S L-7 by adopting a molybdenum-antimony colorimetric resistance method, and measuring an absorbance value at the wavelength of 720nm by using a visible spectrophotometer to quantitatively analyze the content of the soluble phosphorus.

And (3) drawing a phosphorus standard curve, namely adding a standard phosphorus solution with a corresponding volume of 100 mg/L into a volumetric flask, adding 2 drops of 2, 6-dinitrophenol as an indicator, adjusting the pH value by using dilute sulfuric acid and a 10% NaOH solution, adding an aluminum antimony anti-color-developing agent of 5m L, fixing the volume to a scale, enabling the standard phosphorus concentration to be 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/L respectively, shaking up, reacting for 30min at room temperature (about 25 ℃), then carrying out color comparison at 720nm by using an ultraviolet spectrophotometer, and drawing the standard curve according to the result.

The content of soluble phosphorus in the culture solution is measured by inoculating S L-7 into a 50m L organophosphorus (or inorganic phosphorus) liquid culture medium, using no inoculation as a control, the rotating speed of a shaking table is 150r/min, culturing at 28 ℃ for 3d, transferring the culture solution into a sterile 50m L centrifuge tube, carrying out ultrasonic cell disruption by using a numerical control ultrasonic cleaner, disrupting for 20min to release available phosphorus in cells, centrifuging at 4000r/min for 20min, taking 2.5m L supernatant, adding 2 drops of 2, 6-dinitrophenol as an indicator into a 50m L bottle, adding a drop of dilute sulfuric acid until the reaction solution is colorless, adding a molybdenum-antimony anti-chromogenic agent 5m L, fixing the volume, reacting, measuring the OD value of the supernatant at 720nm by using an ultraviolet spectrophotometer, obtaining the content of the available phosphorus in the supernatant according to a standard curve, and obtaining the results as shown in Table 1:

TABLE 1 test results of phosphorus solubilizing ability

As can be seen from FIG. 3 and Table 1, the strain S L-7 showed significant phosphate-solubilizing rings on both inorganic phosphorus and organic phosphorus medium plates, so that the strain S L-7 was preliminarily judged to have certain phosphate-solubilizing ability.

Example 4

In order to determine the influence of oxygen content on the enzyme production of the strain S L-7, the rotation speeds of a shaking table are respectively set to be 140rpm, 160rpm, 180rpm, 200rpm and 220rpm, the strain is inoculated in a 100m L fermentation enzyme production culture medium according to 4 percent of inoculum size (4 m L bacterial suspension is inoculated in every 100m L CMC fermentation enzyme production culture medium), the shaking table is subjected to shaking culture at 37 ℃, and CMCase and FPase are determined on the 3 rd day, the result is shown in figure 4, the enzyme production activity of the strain is in a trend of increasing and then tending to balance along with the increase of the rotation speed of fermentation liquid, the FPase and the CMCase are highest when the rotation speed of the fermentation liquid is 200 and are 40.93U/m L and 106.56U/m L, and in conclusion, the optimal enzyme production rotation speed of the strain is 200rpm, and the optimal enzyme production rotation speed range is 180-200 rpm.

Example 5

In order to determine the influence of temperature on the enzyme production of the strain S L-7, the table temperature of a shaking table is respectively set to be 28 ℃, 37 ℃, 46 ℃, 55 ℃ and 60 ℃, the shaking table is inoculated into a 100m L fermentation enzyme production culture medium according to the 4% inoculation amount (4 m L bacterial suspension is inoculated into every 100m L CMC fermentation enzyme production culture medium), the shaking table is shaken and cultured under the condition of the rotation speed of 200rpm, and the CMCase and FPase are determined on the 3 rd day, the result is shown in figure 5, the enzyme production activity of the strain is in the change trend of firstly rising and then falling and finally tending to balance along with the increase of the temperature of fermentation liquor, the FPase and the CMCase are the highest at the temperature of 37 ℃, and are 39.09U/m L and 108.86U/m L, and in conclusion, the optimum enzyme production temperature range of the strain is 37 ℃ and the optimum enzyme production temperature range is 25-45.

Example 6

The inoculation amounts are respectively set to be 2%, 4%, 6%, 8%, 10% and 12% (2 m L, 4m L, 6m L, 8m L, 10m L and 12m L bacterial suspensions are respectively inoculated into every 100m L CMC fermentation enzyme production culture medium), the strains are inoculated into 100m L CMC fermentation enzyme production culture medium, shaking table shaking culture is carried out at 37 ℃ and 200rpm, CMCase and FPase are respectively measured on the 3 rd day, the results are shown in figure 6, the enzyme production activity of the strains shows a change trend of rising firstly and then falling and finally tending to balance along with the increase of the inoculation volume, when the inoculation volume is 8%, the maximum values of the FPase and the CMCase are 40.94U/m L and 115.96U/m L, and in conclusion, the optimal enzyme production inoculation volume of the strains is 8%, and the optimal enzyme production inoculation volume range is 6-10%.

Example 7

Inoculating the bacterial suspension into a fermentation enzyme production culture medium of 100m L according to the inoculation amount of 8%, adjusting the initial pH of the fermentation enzyme production culture medium to 5.0, 6.0, 7.0, 8.0 and 9.0 respectively, carrying out shaking culture at 37 ℃ and 200rpm, and measuring the CMCase and the FPase on the 3 rd day, wherein the results are shown in figure 7, as the pH of the fermentation liquor is increased, the enzyme production activity of the bacterial strain shows a change trend of firstly rising and then falling and finally tending to balance, when the pH of the fermentation liquor is 7, the CMCase and the FPase are highest and are 114.31U/m L and 54.57U/m L, in conclusion, the optimal enzyme production pH of the bacterial strain is 7, and the optimal enzyme production pH range is 6-8.

Example 8

In order to determine the effect of different nitrogen sources on the enzyme production of the strain, 0.4% peptone, 0.4% ammonium sulfate and 0.4% urea were respectively set as nitrogen sources, and the bacterial suspensions were inoculated into 100m L CMC fermentation enzyme production medium at 8% inoculum size, shake cultivation was carried out at 37 ℃ and 200rpm, and the results of CMCase and FPase were respectively determined on day 3 as shown in FIG. 8. in the peptone treatment, CMCase and FPase could respectively reach 119.1U/m L and 75.22U/m L. in the urea treatment, CMCase and FPase could respectively reach 59.73U/m L and 9.22U/m L. in the ammonium sulfate treatment, CMCase and FPase could respectively reach 75.96/m L and 15.87U/m L. in the peptone treatment, CMe and FPase were higher than urea and ammonium sulfate, whereby peptone promoted the enzyme production of the strain, and urea and ammonium sulfate produced different degrees of inhibition of the enzyme production.

Example 9

In order to determine the influence of different carbon sources on the enzyme production of the strain, the carbon sources are respectively set to be 0.5 percent of glucose, 0.5 percent of CMC-Na and 1 percent of tobacco straw powder (0.5 percent of soluble carbon source or l percent of insoluble carbon source), the bacterial suspension is inoculated into a fermentation enzyme production culture medium of 100m L CMC in an inoculation amount of 8 percent, shaking culture is carried out at 37 ℃ and 200rpm, and the results of CMCase and FPase are respectively determined on the 3 rd day, as shown in the figure 9, in the CMC-Na treatment, the CMCase and the FPase can respectively reach 123.89U/m L and 90.71U/m L, in the tobacco straw powder treatment, the CMCase and the FPase can respectively reach 105.46U/m L and 53.84U/m L, in the glucose treatment, the Case and the FPase can respectively reach 59.73U/m L and 38.35U/m L, and in the CMCase-Na treatment, the tobacco straw powder and the tobacco straw powder can respectively inhibit the enzyme production of the CMC-Na, and the strain can promote the action of the CMC-Na.

Example 10

To determine the effect of different carbon nitrogen ratios on the enzyme production of the strains, the optimal carbon source and the optimal nitrogen source optimized in the above experiment were selected, the carbon nitrogen ratios were set to be 2:1, 1:1 and 1:2, respectively, and the bacterial suspension was inoculated into a 100m L fermentation enzyme production medium at 8% inoculum concentration, shake cultivation was performed at 37 ℃ and 200rpm, and CMCase and FPase were determined on day 3, as shown in FIG. 10, the C/N ratio was 2:1, the CMCase and FPase could respectively reach 135.69U/m L, 67.85U/m L, the C/N ratio was 1:1, the CMCase and FPase could respectively reach 120.21U/m L, 87.02U/m L, the C/N ratio was 1:2, the CMe and FPase could respectively reach 84.07U/m L, 103.98U/m L, the C/N ratio was 2:1, the CMe and FPase could respectively reach 84.1: 1, and the C/N ratio was higher than the C/N ratio was 1:1, thereby the effect on the enzyme production was observed.

Example 11

1. Construction of tobacco straw high-efficiency microbial inoculum

Inoculating S L-7 into peptone as a unique nitrogen source in a culture medium, CMC-Na as a unique carbon source in the culture medium and CMC (carbon/nitrogen) with the C/N ratio of 2:1, culturing for three days under the conditions of optimum enzyme-producing culture environment, pH7.0, inoculation volume of 8%, rotation speed of 200rpm and temperature of 37 ℃, and constructing the tobacco straw high-efficiency degradation microbial inoculum.

2. Composting tobacco straw compost

2.1 addition of microbial inoculum

The microbial inoculum was added to the watering can. Then spreading the tobacco straw compost raw material, and then mixing the microbial inoculum with the tobacco straw compost raw material 5:100 mass percent of the fertilizer is uniformly sprayed on the surface of the spread tobacco straw compost raw material. After spraying, stirring uniformly, adjusting the water content of the mixture to 50% -60%, and starting composting.

2.2 Stacking

The compost raw materials added with the microbial inoculum are filled into a simple composting device and are piled into a pile body with the height of about 0.56m and the volume of about 60L, the temperature of the pile body is regularly monitored every day in the piling process, a temperature measuring point is positioned in the middle of the pile body, natural ventilation is carried out, the pile body enters a front decomposition stage when the temperature of the pile body rises to about 70 ℃, the pile body is turned over once every 4-5 days until the temperature of the pile body drops to 50 ℃ or below, the pile body is turned over once every 2 days until the temperature of the pile body drops to below 40 ℃ and does not rise, the pile body stops turning over, the pile body enters a rear decomposition stage, the mass ratio of carbon elements and nitrogen elements in the pile body is regularly monitored every day, when the mass ratio of the carbon elements and the nitrogen elements is less than 20, the rear decomposition stage is finished, and the whole piling process is also finished.

The invention provides a bacillus amyloliquefaciens strain S L-7, wherein the strain S L-7 has strong capacity of degrading cellulose and dissolving phosphorus and can be used for preparing tobacco straw compost.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> college of teachers and universities on Fuyang

<120> bacillus amyloliquefaciens S L-7 and application thereof

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tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc 420

aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480

gccgcggtaa tacgtaggtg gcaagcgttg tccggaattattgggcgtaa 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

1. A strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) S L-7 with the preservation number of GDMCC No. 60630.

2. The bacillus amyloliquefaciens S L-7 according to claim 1, wherein the 16S gene sequence of the bacillus amyloliquefaciens S L-7 is shown as SEQ ID No. 1.

3. Use of the bacillus amyloliquefaciens S L-7 of claim 1 or 2 for degrading cellulose.

4. Use of the bacillus amyloliquefaciens S L-7 of claim 1 or 2 for phosphate solubilization.

5. The application of the bacillus amyloliquefaciens S L-7 in the preparation of a tobacco straw degrading microbial inoculum according to claim 1 or 2.

6. The application of the bacillus amyloliquefaciens S L-7 as the main component of the tobacco straw degrading microbial inoculum according to claim 5, wherein the preparation method of the tobacco straw degrading microbial inoculum comprises the following steps of inoculating the bacillus amyloliquefaciens S L-7 into a CMC fermentation culture medium to be cultured for 3d, and obtaining the tobacco straw degrading microbial inoculum, wherein peptone is used as a unique nitrogen source, CMC-Na is used as a unique carbon source, and the C/N ratio of the unique carbon source to the unique nitrogen source is 2: 1.

7. The use of claim 6, wherein the inoculation volume of the bacillus amyloliquefaciens S L-7 is 6-10% of the volume of the CMC fermentation medium.

8. The use according to claim 6, wherein the temperature of the culture is 25-45 ℃ and the pH value is 6.0-8.0.

9. The use according to claim 8, wherein the culturing is accompanied by stirring, and the stirring speed is 180-200 rpm.