CN106957807B

CN106957807B - Bacillus licheniformis strain TA65 and application thereof in promoting compost maturity

Info

Publication number: CN106957807B
Application number: CN201710184427.5A
Authority: CN
Inventors: 李群良; 陆彦宇; 徐佳琦
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2017-03-24
Filing date: 2017-03-24
Publication date: 2020-07-28
Anticipated expiration: 2037-03-24
Also published as: CN106957807A

Abstract

The invention belongs to the technical field of development of plant growth-promoting bacteria, and particularly relates to a bacillus licheniformis strain TA65 and application thereof in promoting compost maturity. A Bacillus licheniformis strain TA65 is classified and named as Bacillus licheniformis TA65(Bacillus licheniformis), and is preserved in China general microbiological culture Collection center (CGMCC) at 2017, 01-05, with a preservation number of CGMCC No. 13531. The bacillus licheniformis strain TA65 is obtained by screening and separating a compost high-temperature period sample for the first time, is high-temperature resistant, can increase and propagate quickly, and can produce lignocellulose degrading enzyme; the microbial inoculum containing the bacillus licheniformis strain TA65 can increase the composting temperature, accelerate the degradation of organic matters and water-soluble organic matters (DOM), increase the content of Kjeldahl nitrogen and promote compost maturity; also has the function of generating biosurfactant, so that the strain can be used for preparing the biosurfactant.

Description

Bacillus licheniformis strain TA65 and application thereof in promoting compost maturity

Technical Field

The invention belongs to the technical field of development of plant growth-promoting bacteria, and particularly relates to a bacillus licheniformis strain TA65 and application thereof in promoting compost maturity.

Background

The fertilizer plays a great role in modern agricultural production. But with the increase of the using amount of the fertilizer, the utilization rate of the fertilizer is reduced year by year. Pesticide and chemical fertilizer residues release a large amount of harmful substances to the environment, pollute soil, water sources and food, pose great threat to human health and living environment, and increasingly strong call for protecting ecological environment and producing safe food is required. Therefore, it has been an urgent necessity to develop a new fertilizer source using an alternative chemical fertilizer to meet the need for developing green agriculture and green food. The microbial fertilizer can make ineffective nutrition in soil effective, prevent and control crop diseases, reduce the use of pesticides and chemical fertilizers, is a fundamental way for solving the pollution of soil, water sources and foods, and is generally considered to be an environment-friendly, economic and effective method for improving the crop yield.

The microbial fertilizer is a specific product containing a microbial living body, is applied to agricultural production and can obtain a specific fertilizer effect. Wherein living microorganisms in the preparation play a critical role. Currently, microbial fertilizer products are generally divided into two main categories: one is a microbial fertilizer in the narrow sense, which means that the supply of plant nutrient elements, including the total supply of plant nutrient elements in soil and production environment, is increased by the life activities of microorganisms, resulting in improvement of the plant nutrient status and further increase of yield. Representative of this class of microbial fertilizers are rhizobia fertilizers; the other is a broad-sense microbial fertilizer, which means that the supply of plant nutrient elements can be improved through the life activities of microorganisms in the fertilizer, plant growth hormone can be generated, the absorption and utilization of the nutrient elements by plants are promoted, or the pathogenic effect of certain pathogenic microorganisms is antagonized, the crop pest and disease damage is reduced, and the crop yield is indirectly improved. Compared with chemical fertilizers, the microbial fertilizer has the following advantages: the soil structure is not damaged; the ecological environment is protected, the environment is not polluted, and the fertilizer is non-toxic and harmless to people and livestock; the fertilizer efficiency is durable; the yield of the crops is improved, and the quality of the crops is improved; low cost, economy and effectiveness.

Plant growth-promoting rhizobacteria (PGPR) is a kind of microorganism capable of high-density colonizing in Plant rhizosphere, and has the functions of inhibiting Plant pathogenic bacteria and harmful microorganism in rhizosphere, promoting Plant growth and increasing crop yield. The research and application of PGPR has played a very important role as an important resource pool for biofertilizers and biopesticides. The research and development of the microbial fertilizer from the aspect of resource recycling are of more realistic significance to the comprehensive utilization of resources and the environmental protection.

Bacillus licheniformis (Bacillus licheniformis) with rod-shaped and single-grown cell morphology and arrangement can regulate dysbacteriosis to achieve therapeutic purpose, and can promote organism to produce antibacterial active substance and kill pathogenic bacteria. Can produce active resisting matter, has unique biological oxygen-taking action mechanism and can inhibit the growth and propagation of pathogenic bacteria. The bacillus licheniformis is mainly applied to the following aspects: (1) effectively prevent enteritis, gill rot and other diseases of aquatic animals. (2) The toxic and harmful substances in the culture pond are decomposed, and the water quality is purified. (3) Has stronger activity of protease, lipase and amylase, promotes the degradation of nutrients in the feed, and ensures that aquatic animals can absorb and utilize the feed more fully. (4) Stimulating the development of immune organs of aquatic animals and enhancing the immunity of organisms. (5) Promoting the growth of normal physiological anaerobic bacteria in the intestinal tract, adjusting the imbalance of intestinal flora and recovering the intestinal function; (6) the medicine has special effect on intestinal bacterial infection, and has obvious curative effect on light or heavy acute enteritis, light and common acute bacillary dysentery and the like; (7) can produce active resisting matter, has unique biological oxygen-taking action mechanism and can inhibit the growth and propagation of pathogenic bacteria.

At present, the bacillus licheniformis is mainly used as an animal feed additive, and no relevant report is found on the application of the bacillus licheniformis in promoting compost maturity.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide a bacillus licheniformis strain TA65 which can be applied to preparing a surfactant and promoting compost maturity.

The purpose of the invention is realized by the following technical scheme:

the invention discloses a Bacillus licheniformis strain TA65 which is classified and named as Bacillus licheniformis TA65(Bacillus licheniformis), and is preserved in China general microbiological culture Collection center (CGMCC) in 2017, 01-05 and with the preservation number of CGMCC No. 13531.

The invention also provides application of the bacillus licheniformis strain TA65 in degradation of lignocellulose.

The invention also provides application of the bacillus licheniformis strain TA65 in preparing a surfactant.

The invention also provides application of the bacillus licheniformis strain TA65 in preparation of laccase, lignin peroxidase, manganese peroxidase, cellulase and hemicellulase.

The invention also provides a microbial inoculum, which is prepared by inoculating the bacillus licheniformis strain TA65 seed solution of claim 1 into L B culture solution after 1% (v/v) sterilization of 1L, culturing for 24h in a shaking table (50 ℃, 120rmp) and collecting the culture solution.

The invention also provides application of the microbial inoculum in promoting compost maturity.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the bacillus licheniformis strain TA65 provided by the invention is obtained by screening and separating a compost high-temperature-stage sample for the first time, is high-temperature-resistant, grows and breeds quickly, and can produce lignocellulose degrading enzyme.

2. The microbial inoculum containing the bacillus licheniformis strain TA65 provided by the invention can increase the composting temperature, accelerate the degradation of organic matters to generate micromolecular water-soluble organic matters (DOM), increase the content of Kjeldahl nitrogen and promote compost maturity.

3. The bacillus licheniformis strain TA65 provided by the invention also has the function of producing a biosurfactant, so that the strain can be used for preparing the biosurfactant.

Description of preservation information

Bacillus licheniformis TA65(Bacillus licheniformis), with the preservation number of CGMCC No.13531, the preservation date of 2017, 01-05 days, the preservation unit is China general microbiological culture Collection center, the preservation address is Beijing West Lu No.1, North Cheng Yang district, 3, institute of microbiology, China academy of sciences.

Drawings

FIG. 1 is a graph of temperature versus composting time. RT: room temperature, blank group (CK), Experimental group (T)

FIG. 2 is a graph of pH as a function of composting time. Blank group (CK), Experimental group (T)

FIG. 3 is a graph of moisture as a function of composting time. Blank group (CK), Experimental group (T)

Figure 4 is a plot of organic matter content as a function of composting time. Blank group (CK), Experimental group (T)

FIG. 5 is a plot of Kjeldahl nitrogen content as a function of composting time. Blank group (CK), Experimental group (T)

FIG. 6 is a graph showing the variation of ammonia nitrogen content with composting time. Blank group (CK), Experimental group (T)

FIG. 7 is a curve of nitrate nitrogen content as a function of composting time. Blank group (CK), Experimental group (T)

FIG. 8 is a graph of DOM content versus composting time. Blank group (CK), Experimental group (T)

FIG. 9 is a colony map of Bacillus licheniformis TA 65.

FIG. 10 is a graph showing the performance of Bacillus licheniformis TA65 on blood agar plates.

FIG. 11 is a glucose standard curve.

FIG. 12 is a xylose standard curve.

FIG. 13 is a gel electrophoresis pattern of the selected strains;

description of the reference numerals:

M-2000bpMake；1-TB21；2-TB22；3-TB23；4-TB24；5-TB25；6-TB41；7-TB42；8-TB43；9-TB44；10-TB45；11-TB61；12-TB62；13-TB63；14-TB64；15-TB65；16-TA65；17-TB46。

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The materials and reagents used in the following examples are commercially available, unless otherwise specified. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Example 1: screening of Bacillus licheniformis TA65(Bacillus licheniformis)

1. Experimental materials and apparatus

1.1 Primary reagents

TABLE 1 Main reagents

1.2 Main instruments and devices

TABLE 2 Main instruments and Equipment

Name of instrument	Model number	Manufacturer of the product
			Ultraviolet-visible spectrophotometer	UV759	Shanghai Jingke
Clean workbench	SW-CJ-1F	SUZHOU ANTAI AIRTECH Co.,Ltd.
			Automatic balance centrifugal machine	PSZ4-1.2	Beijing City medical centrifuge factory
Rafender pH meter	PHS-2S	Shanghai electric Instrument Co Ltd
			Constant temperature shaking table	HQ45Z	Wuhan Zhongke science and technology development liability company
Biochemical incubator	SPX-150B	Shanghai leap-in medical instrument factory
			Electronic temp. -regulating universal electric stove	DK-98-II	TIANJIN TAISITE INSTRUMENT Co.,Ltd.
Refrigerator with a door
			Electronic balance	YP6102	Shanghai Guang medical instruments Ltd
Electric heating drying box
			Vertical pressure steam sterilizer	YXQ-LS-50SII	Shanghai Bowen realty Co Ltd medical equipment factory
Electric heating constant temperature water bath		Beijing medical facilities factory
			Precision electronic balance	JJ500Y

1.3 Medium

The bacteria culture medium comprises 33g of nutrient agar and 1000m of deionized water L;

l B culture solution, 10g peptone, 5g yeast powder, 10g sodium chloride and 1000m L deionized water.

2. Separation and screening of lignin-degrading bacteria

2.1 sampling

The screened samples are compost samples of 2 days, 4 days and 6 days respectively, the compost samples are sampled at multiple points (generally 10-20cm below the surface of the compost) at a higher temperature of the compost, and the samples are uniformly mixed and then put into a sample bag to be stored in a refrigerator at the temperature of-4 ℃.

2.2 screening and purification of thermophilic bacteria

Weighing 10g of sample, adding into a triangular flask containing 90m L sterilized NaCl solution (0.9% w/v), placing on a shaking table, oscillating for 30min to break loose zoogloea and disperse bacteria in the solution in a single cell state, adding sterilized NaCl solution into 7 test tubes (sterilized in the same way), adding 9m L into each test tube, adding vibrated bacteria solution 1m L into the test tubes, respectively, namely diluting to 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7Then 0.1m L of the bacterial liquid at each dilution is coated on a bacterial culture medium plate and cultured at a constant temperature of 50 ℃, each plate with dilution gradient is parallel to 5 plates, and the bacterial colony can be observed to fall at 10^-5The growth vigor is better under the dilution degree. Larger colonies were selected in five parallel plates for isolation and streaking for multiple purifications.

As shown in FIGS. 9-10, the colony morphology of Bacillus licheniformis TA65 is regular, the colony size is small, the surface is rough and dry, the edge is wet, the color is white, and the colony edge is regular.

3. Preparation of crude enzyme solution

L B culture solution is prepared, sterilized, poured into a serum bottle (25m L) sterilized in the same way, the strain obtained by separation is inoculated into the serum bottle, and is put into a shaking table (50 ℃, 120rmp) for culture to prepare a seed solution L B culture solution is prepared, 100m L is poured into a conical bottle for sterilization, the 1m L seed solution is absorbed into the conical bottle, and is also put into the shaking table for culture (50 ℃, 120rmp) to prepare a bacterial solution.

Liquid enzyme production culture, centrifuging the bacterial solution at 5000r/mim for 5min, collecting supernatant as crude enzyme solution, and using heat inactivated enzyme solution (0.6 m L crude enzyme solution in centrifuge tube, boiling for 10min) as control.

4. Method for measuring enzyme activity

4.1 determination of laccase (L ac) Activity

1. Reagent

(1) Determined by the rate at which the enzyme oxidizes ABTS;

(2)0.1mo L/L citric acid-sodium citrate buffer, which is prepared by weighing 2.941g of sodium citrate and 2.1014g of citric acid respectively, adding deionized water 100m L, adding citric acid into sodium citrate, and adjusting the pH value to 5.0 by using a pH meter;

(3)0.5mL 0.5moL/L ABTS；

2. procedure for the preparation of the

(1) At 25 ℃, 2m L citric acid-sodium citrate buffer (0.1mmo L/L, pH 5), and 0.5m L ABTS were added to the tube, and the mixture was poured into a cuvette;

(2) then, 1m L enzyme solution was added to start the reaction, and the change in absorbance was measured at 420 nm.

3. Calculation method

The enzyme activity is defined as that ABTS which oxidizes 1 mu mo L per minute is an enzyme activity unit,

extinction coefficient 3.6 x 10⁴[(moL/L)^-1cm^-1]Enzyme activity unit U/L

Laccase (L ac) activity Δ OD 10⁶/ΔT*V_{Enzyme solution}*∈

＝(ΔOD/ΔT)*10⁶/0.5*3.6*10⁴

In the formula: Δ OD-the value at which the difference between adjacent absorbance values measured is greatest; the time difference value corresponding to the delta T-delta OD value;

v-volume of enzyme solution.

4.2 measurement of Lignin peroxidase (L ip) Activity

1. Reagent

(1)0.24mo L/L sodium tartrate buffer solution, which is prepared by respectively weighing 3.60216g of tartaric acid and 5.52192g of sodium tartrate, adding deionized water 100m L, adding tartaric acid into the sodium tartrate, and adjusting the pH to 3.0;

(2)0.1m L24 mmo L/L veratryl alcohol;

(3)0.05m L6.0.6.0 mm o L/L H₂O₂；

2. Procedure for the preparation of the

(1) At 37 ℃, the total reaction system was 3m L, 1.85m L0.24 mo L/L sodium tartrate buffer (pH 3.0), and 0.1m L24 mmo L/L veratryl alcohol and 1.0m L enzyme solution were added to the tube;

(2) preheating to 37 deg.C, pouring the solution into a cuvette, and adding 0.05m L6.0.0 mm H L/L₂O₂The reaction was started and the change in absorbance was measured at a wavelength of 310 nm.

3. Calculation method

The enzyme activity is defined as that veratryl alcohol which oxidizes 1 mu mo L per minute is an enzyme activity unit,

extinction coefficient 9.3 x 10³[(moL/L)^-1cm^-1]Enzyme activity unit U/L

Peroxidase (L ip) activity Δ OD 10⁶/ΔT*V_{Enzyme solution}*∈

＝(ΔOD/ΔT)*10⁶/1*9.3*10³

v-volume of enzyme solution.

4.3 determination of manganese peroxidase (Mnp) Activity

1. Reagent

(1)0.11mo L/L sodium lactate buffer, weighing 1.10098g lactic acid and 2.05516g sodium lactate, adding deionized water 100m L respectively, adding lactic acid into sodium lactate, and adjusting the pH to 4.5;

(2)0.025M l40 mmo L/L manganese sulfate;

(3)0.025m L1.6 mm o L/L H₂O₂；

2. Procedure for the preparation of the

(1) According to the enzyme in H₂O₂In the presence of Mn²⁺Oxidized Mn³⁺Is determined by the speed of the motor;

(2) the total reaction volume was 1m L, 0.85m L0.11.11 mo L/L sodium lactate buffer (pH 4.5) was added to the tube, and 0.025m L, 40mmo L/L MnSO were added₄And 1m L of enzyme solution;

(3) after preheating at 37 ℃ the solution was poured into a cuvette to which 0.025m L1.6.6 mm H L/L was added₂O₂The reaction was started and the change in absorbance was measured at 240 nm.

3. Calculation method

Enzyme activity is defined as the oxidation of 1. mu. mo L Mn per minute²⁺Is Mn³⁺Is an enzyme activity unit, and is characterized in that,

extinction coefficient 6.5 x 10³[(moL/L)^-1cm^-1]Enzyme activity unit U/L

Manganese peroxidase (Mnp) activity Δ OD 10⁶/ΔT*V_{Enzyme solution}*∈

＝(ΔOD/ΔT)*10⁶/1*6.5*10³

v-volume of enzyme solution.

4.4 determination of cellulase Activity

1. Reagent

(1)0.1mo L/L sodium citrate-sodium citrate buffer, which is prepared by weighing 2.941g of sodium citrate and 2.1014g of citric acid respectively, adding deionized water 100m L, adding citric acid into sodium citrate, and adjusting the pH value to 4.8 by using a pH meter;

(2)1.5mLDNS；

(3) glucose standard solution 1.0000g glucose is dissolved in 1000ml deionized water to prepare 1mg/m L glucose standard solution.

2. Standard glucose Curve preparation

Taking 1mg/m L standard glucose solution 0,0.2,0.4,0.6,0.8,1.0 and 1.2m L respectively in a test tube, adding deionized water to 2.0m L, adding 2.0m L DNS reagent, plugging, carrying out boiling water bath for 10min, cooling, fixing the volume to 15m L, measuring an OD value by using a spectrophotometer at the wavelength of 550nm, repeating the experiment for 3 times, taking a mean value to draw a graph, and taking a glucose standard curve as shown in figure 11.

3. Procedure for the preparation of the

(1) Adding Whatman NO1 quantitative test paper of 1cm × 2cm into the test tube, and adding 1.0cm sodium citrate-sodium citrate buffer (0.1mo L/L, pH 4.8) and 0.5m L enzyme solution;

(2) preserving heat at 50 ℃ for 1h, taking out, adding 1.5m L DNS to terminate the reaction, carrying out water bath for 5min, cooling with running water, and metering the volume to 25m L;

(3) the absorbance was measured at 540 nm.

4. Calculation method

Cellulase activity ═ y 1000ug/V_{Enzyme solution}T, where y is derived from the standard 0.6916x

In the formula: x-measured absorbance values; t-time of water bath; v_{Enzyme solution}Volume of enzyme solution.

4.5 hemicellulase Activity assay

1. Reagent

(1)1.8m L1% xylan solution prepared into 1% solution with xylan in acetic acid buffer solution with pH 4.8;

(2)1.8m L acetic acid buffer solution, weighing 0.82g sodium acetate, weighing 0.6m L acetic acid, respectively adding deionized water 100m L, adding acetic acid into the sodium acetate, and adjusting the pH value to 4.8;

(3)2mL DNS；

(4) 1.0000g of xylose standard solution, namely dissolving xylose in 1000m of L deionized water to prepare 1mg/m of L xylose standard solution.

2. Production of xylose curve

Taking standard xylose liquid of 1mg/ml, 0,0.2,0.4,0.6,0.8,1.0 and 1.2m L respectively, adding deionized water to 2.0m L, then adding 2.0m L DNS reagent, plugging and boiling water bath for 10min, cooling, fixing the volume to 15m L, measuring OD value with a spectrophotometer at the wavelength of 550nm, repeating the test for 3 times, taking the mean value to draw a graph, and taking a xylose standard curve as shown in figure 12.

3. Procedure for the preparation of the

(1) Adding enzyme solution 0.2m L into a 15m L graduated test tube, and absorbing xylan solution 1.8m L1% respectively;

(2) adding 0.2m L enzyme solution into 1.8m L acetic acid buffer solution without adding xylan solution, and shaking up;

(3) taking out after 60min of 50 ℃ water bath, absorbing 2m L DNS reagent, shaking up, plugging, immediately boiling water bath for 10min, cooling, adding water to constant volume of 15m L, and shaking up and down gently;

(4) the absorbance was measured at a wavelength of 550nm while adjusting the point to zero with a blank.

4. Calculation method

Calculating according to the formula: hemicellulase activity ═ y 1000ug/V_{Enzyme solution}(ii) T, wherein y is 1.1504x +0.008 from standard; in the formula: x-measured absorbance values; t-time of water bath; v_{Enzyme solution}Volume of enzyme solution.

5. Results

TABLE 3 determination of the enzyme-producing Activity of Bacillus licheniformis TA65 Strain

As shown in Table 3, Bacillus licheniformis TA65 produced cellulase with the highest activity of 851U/L, followed by manganese peroxidase with the activity of 264U/L, while laccase, lignin peroxidase and hemicellulase had activities of 23U/L, 44U/L and 16U/L, respectively.

Example 2: identification of Bacillus licheniformis TA65(Bacillus licheniformis)

2.1 Main Instrument of experiment

TABLE 4 Instrument for experiments

2.2 Experimental procedures

2.2.1 extraction of DNA

DNA was extracted using the "Ezup column type bacterial genome DNA extraction kit" of the Producer. The specific operation steps are detailed in the specification.

2.2.2PCR amplification

PCR amplification was performed using 2 × Es Taq MasterMix, and the reaction system was as follows:

reaction procedure:

2.2.3 electrophoresis results of PCR products

Using 1% agarose gel electrophoresis, voltage 120V, electrophoresis for 30min, each hole loading 1u L, the electrophoretogram is shown in FIG. 13.

As shown in FIG. 13, the electrophoresis results of the PCR products showed that the 16S rDNA fragment of the bacterium was successfully amplified.

2.2.4 sequencing and alignment results

Sequencing Using primers 27F/1492R, the sequence of Bacillus licheniformis strain TA65 is shown in SEQ.1.

B L ASTN comparison was performed in NCBI gene bank to obtain the strain identification results, and the results are shown in Table 5.

TABLE 5 Bacillus licheniformis strain TA65 identification data

As shown in Table 5, strain TA65 showed 99% similarity to Bacillus licheniformis, and therefore, the strain was named Bacillus licheniformis TA65(Bacillus licheniformis).

Example 3: application of microbial inoculum containing bacillus licheniformis strain TA65 in promoting compost maturity

3.1 preparation of the bacterial agent

Inoculating TA65 seed solution into L B culture solution after 1% (v/v) sterilization by using the inoculation amount of 1% (v/v), culturing for 24h in a shaking table (50 ℃, 120rmp), collecting the culture solution to obtain the microbial inoculum, adding the microbial inoculum to obtain an experimental group (T), and adding L B culture solution after 1L sterilization to a blank group (CK).

3.2 compost test

The compost raw materials are cow dung and sugarcane leaves, and the mass ratio is 17:3, and the total weight is 20 kg. And (4) composting for 45d, sampling at 0 th, 5 th, 10 th, 16 th, 23 th, 30 th and 45 th d, and measuring parameters such as temperature, pH, water content, organic matters, Kjeldahl nitrogen, inorganic nitrogen, DOM content and the like. The microbial inoculum is inoculated twice in the whole composting process, wherein the inoculation time is 0d and 10d respectively. The stack was turned three times for 10d, 20d and 30d respectively.

3.3 results of the experiment

The Bacillus licheniformis TA65 has high temperature resistance, fast propagation and capacity of producing lignocellulose degrading enzyme.

Compared with Compost (CK) without added microbial inoculum, compost (T) with added TA65 microbial inoculum has the following advantages:

(1) the temperature of the composting process is that the temperature of the heap with TA65 microbial inoculum is higher than that of the heap without the microbial inoculum. Is beneficial to killing pathogens in the compost, accelerating the high-temperature degradation of compost materials and promoting the compost to be thoroughly decomposed (figure 1).

(2) As can be seen from FIG. 2 and FIG. 3, the pH of the compost to which the TA65 microbial inoculum is added is increased higher, and the water content is reduced faster, that is, the reaction of the compost to which the microbial inoculum is added is more violent, so that NH is caused₃The large amount of volatilization increases the pH value, generates more heat and takes away a large amount of moisture.

(3) From the degradation rate of Organic Matters (OM), the OM without adding the microbial inoculum is reduced from 90.38% to 78.67, the degradation rate is 12.95%, the OM with adding the TA65 microbial inoculum is reduced from 90.00% to 76.62%, and the degradation rate is 14.87% (fig. 4).

(4) From the viewpoint of the change of the content of the heap soluble organic matter (DOM), the DOM content without adding the microbial inoculum is reduced from 69.6mg/g to 9.8mg/g by 85.9%, and the DOM content with adding the TA65 microbial inoculum is reduced from 75.6mg/g to 9.7mg/g by 87.2% (FIG. 8).

In conclusion, the TA65 microbial inoculum is added, so that the composting temperature can be increased, the degradation of organic matters and DOM is accelerated, the content of Kjeldahl nitrogen is increased, and the compost is promoted to be thoroughly decomposed.

Example 4: surfactant production by Bacillus licheniformis strain TA65

The surfactant-producing status of Bacillus licheniformis strain TA65 was identified by blood agar plate and surface tension meter.

The blood agar plate is prepared by sterilizing nutrient agar (Beijing Luqiao), cooling to about 50 deg.C, adding sterilized defibered sheep blood 5-10m L per 100m L nutrient agar in sterile environment, shaking, pouring into plate, and making into slant.

And (3) measuring the surface tension of the fermentation liquor: the fermentation liquor surface tension is measured by adopting a ring method, and a full-automatic surface tension meter and an instrument model are adopted: BZY-1.

Experiments have shown that Bacillus licheniformis strain TA65 produces a surfactant.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

SEQUENCE LISTING

<110> Guangxi university

<120> Bacillus licheniformis strain TA65 and application thereof in promoting compost maturity

<130>ZYWS

<160>1

<170>PatentIn version 3.3

<210>1

<211>1450

<212>DNA

<213> Artificial sequence

<400>1

ccccgggcgc tcctataatg cagtcgagcg gaccgacggg agcttgctcc cttaggtcag 60

cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 120

accggggcta ataccggatg cttgtttgaa ccgcatggtt caaacataaa aggtggcttt 180

tcgctaccac ttacagatgg acccgcggcg cattagctag ttggtggggt aacggctcac 240

caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300

gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360

gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa aactctgttg ttagggaaga 420

acaagtaccgttcgaacagg gcggtacctt gacggtacct aaccagaaag ccacggctaa 480

ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540

taaagcgcgc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag 600

ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660

gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 720

tgacgctgag gcgcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 780

cgtaaacgat gagtgctaag tgttagaggg tttccgccct ttagtgctgc agcaaacgca 840

ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 900

cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960

cttgacatcc tctgacaacc ctagagatag ggcttcccct tcgggggcag agtgacaggt 1020

ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080

aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 1140

accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200

gtgctacaat gggcagaaca aagggcagcg aagccgcgag gctaagccaa tcccacaaat 1260

ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 1320

tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380

ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct ttggagccag ccgccgaagg 1440

tgatcagagt 1450

Claims

1. A Bacillus licheniformis strain TA65 is classified and named as Bacillus licheniformis (Bacillus licheniformis), and is preserved in China general microbiological culture Collection center (CGMCC) at 2017, 01-05, with a preservation number of CGMCC No. 13531.

2. Use of bacillus licheniformis strain TA65 according to claim 1 for degrading lignocellulose.

3. Use of bacillus licheniformis strain TA65 according to claim 1 for the preparation of surfactants.

4. Use of bacillus licheniformis strain TA65 according to claim 1 for the preparation of laccase, lignin peroxidase, manganese peroxidase, cellulase and hemicellulase.

5. A microbial inoculum is characterized in that the bacillus licheniformis strain TA65 seed solution of claim 1 is inoculated into L B culture solution after 1% (v/v) sterilization in a shaking table according to the inoculation amount of 1%, the culture solution is cultured for 24h in the shaking table, the rotation speed of the shaking table is 120rpm, the temperature of the shaking table is 50 ℃, and the culture solution is collected, thus obtaining the microbial inoculum.

6. Use of a microbial inoculum according to claim 5 in promoting compost maturity.