CN107058177B

CN107058177B - Geobacillus thermosyphus denitrificus strain TB62 and application thereof in promoting compost maturity

Info

Publication number: CN107058177B
Application number: CN201710183284.6A
Authority: CN
Inventors: 李群良; 徐佳琦; 陆彦宇
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: CN107058177A

Abstract

The invention belongs to the technical field of development of plant growth-promoting bacteria, and particularly relates to a geobacillus thermosiphonicola strain TB62 and application thereof in promotion of compost maturity. A Geobacillus thermosiphonii strain TB62 is classified and named as Geobacillus thermosiphonii TB62(Geobacillus thermosiphonificans) and is preserved in China general microbiological culture Collection center (CGMCC) in 2017, 01-05.. The thermal denitrifying geobacillus strain TB62 is obtained by screening and separating from a compost high-temperature period sample for the first time, is high-temperature resistant, can increase and reproduce quickly, and can produce lignocellulose degrading enzyme; the microbial inoculum containing the ureaplasma urealyticum strain TB62 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

Geobacillus thermosyphus denitrificus strain TB62 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 geobacillus thermosiphonicola strain TB62 and application thereof in promotion of 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 increased 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 introduction of plant diseases and insect pests is reduced, and the crop yield is increased. 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 belongs to the family Bacillaceae, the genus Bacillus, gram-positive bacilli capable of forming endospore-spores, aerobic or facultative aerobic, with flagella. The bacillus is widely distributed in soil, air, water and animal intestinal tracts, has strong stability, and once spores are formed, the bacillus can resist various adverse conditions, such as dry heat (the spores still survive for 1 hour at 150 ℃), moist heat, ultraviolet rays, strong acid, strong base, organic solvent, extreme dryness, vacuum drying, oxidation of oxidant and the like. Under certain conditions, the spores can be stored for a long time, and the effective period of the probiotic product can be ensured. Bacillus is the most desirable microbial additive in all genera.

A large number of tests prove that the bacillus additive can obviously improve the flora composition in organisms and has the effect of promoting growth, and the bacillus has higher stability in the processing process of granules and powder and in an acid environment, can play a role in proliferation in the environment in intestinal tracts and accords with the conditions of microbial additives. The bacillus has high stability in the acidic environment of the intestinal tract, can secrete protease and amylase with stronger activity, and promotes the digestion of nutrient substances of the feed. Bacillus can reduce the number of Escherichia coli in feces and digestive tract.

Currently, the Bacillus strains mainly used include Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, Bacillus toyoi and the like, and researches on geobacillus thermosiphonifer are rarely reported, for example, an invention patent of application No. 201510455215.7 entitled "geobacillus thermosiphonifer CF-1 and application thereof" discloses a preservation No. CGMCC No.10697 and discloses application of the strain in microbial oil recovery.

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 thermosyphon strain TB62 which can be used for preparing a surfactant and promoting compost maturity.

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

the invention discloses a Geobacillus thermosiphonii strain TB62 which is classified and named as Geobacillus thermosiphonii TB62(Geobacillus thermosiphonificans) and is preserved in China general microbiological culture Collection center (CGMCC) at 2017, 01 and 05, with the preservation number of CGMCC 13530.

The invention also provides application of the geobacillus thermosiphonicola strain TB62 in degradation of lignocellulose.

The invention also provides application of the geobacillus thermosiphonicola strain TB62 in preparation of a surfactant.

The invention also provides application of the geobacillus thermosiphonii strain TB62 in preparation of laccase, lignin peroxidase, manganese peroxidase, cellulase and hemicellulase.

The invention also provides a microbial inoculum, which is prepared by inoculating the seed solution of the geobacillus thermosyphus strain TB62 of claim 1 into L B culture solution after 1L sterilization according to the inoculation amount of 1% (v/v), culturing for 24h in a 120rmp shaking table at 50 ℃, 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 geobacillus thermosiphonii strain TB62 provided by the invention is obtained by screening and separating from a compost high-temperature period 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 geobacillus thermoshrinking strain TB62 can increase the composting temperature, accelerate the degradation of organic matters and DOM, increase the content of Kjeldahl nitrogen and promote the compost to be thoroughly decomposed.

3. The geobacillus thermosiphonii strain TB62 provided by the invention also has the function of generating biosurfactant, so that the strain can be used for preparing the biosurfactant.

Description of preservation information

Geobacillus thermodenitrificans TB62(Geobacillus thermodenitrificans) with the preservation number of CGMCC13530 and the preservation date of 2017, 01 and 05 days, wherein the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Beijing university Hodgkin region, Xilu No.1 of Beijing university, and the institute of microorganisms of 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 shows a colony of Geobacillus thermosulfinitificans TB 62.

FIG. 10 shows the behavior of Geobacillus thermomodenitificas TB62 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 Geobacillus thermosulfinitificans TB62

1. Experimental materials and apparatus

1.1 Primary reagents

TABLE 1 Main reagents

Name of medicine	Molecular formula	Specification of
			ABTS	C₁₈H₂₄N₆O₆S₄	AR
Hydrogen peroxide	H₂O₂	AR
			Citric acid	C₆H₈O₇	AR
Citric acid sodium salt	Na₃C₆H₅O₇	AR
			Glacial acetic acid	CH₃COOH	AR
Anhydrous sodium acetate	CH₃COONa	AR
			Xylose	C₅H₁₀O₅	AR
Xylan	(C₅H₈O₄)_n	AR
			Tartaric acid sodium salt	C₄H₅Na₂O₆	AR
Tartaric acid	C₄H₆O₆	AR
			Veratryl alcohol	C₈H₁₂O₃	AR
Lactic acid	C₃H₆O₃	AR
			Sodium lactate	C₃H₅O₃Na	AR
Manganese sulfate	MnSO₄	AR
			Sodium hydroxide	NaOH	AR
Hydrochloric acid	HCl	AR
			DNS reagent

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-into medical treatmentInstrument 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 of Geobacillus thermodenitrificans TB62 has irregular shape, small size, rough surface, moisture, slight transparency, light yellow color, and irregular edge.

3. Preparation of crude enzyme solution

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

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

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 ═ 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 Geobacillus thermodenitificans TB62 Strain

As shown in Table 3, the activity of manganese peroxidase produced by Geobacillus thermositridovum TB62 was the highest, 136U/L, followed by hemicellulase, whose activity was 27U/L, whereas the activities of laccase, lignin peroxidase and cellulase were 9U/L, 4U/L and 5U/L, respectively.

Example 2: identification of Geobacillus thermosulfinitificans TB62

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.

2.2.2 PCR 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

The sequencing is carried out in a two-way mode by using a primer 27F/1492R, and the sequence of the Geobacillus thermodenitrificans TB62 is shown as 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 Geobacillus thermodenitrificans TB62 identification data

As shown in Table 5, the strain TB62 showed 99% similarity to Geobacillus thermosulfitificians, and thus the strain was named Geobacillus thermosulfinitidus TB62(Geobacillus thermosulfinitidus).

Example 3: application of microbial inoculum containing geobacillus thermoshrinkinicum TB62 in promotion of compost maturity

3.1 preparation of the bacterial agent

Inoculating TB62 seed solution into L B culture solution sterilized by 1% (v/v) according to the inoculation amount of 1%, culturing for 24h in a 120rmp shaking table under the condition of 50 ℃, collecting the culture solution which is the microbial inoculum, adding the microbial inoculum to obtain an experimental group (T), and adding L B culture solution sterilized by 1L 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 geobacillus thermosiphonii TB62 is high temperature resistant, can grow and propagate quickly, and can produce lignocellulose degrading enzyme.

Compared with Compost (CK) without added bacterial agent, compost (T) with added TB62 bacterial agent has the following advantages:

(1) from the temperature of the composting process, the temperature of the heap with the added TB62 microbial inoculum is higher than that of the heap without the added 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 TB62 microbial inoculum is added is increased higher, and the water content is reduced faster, namely, 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), OM without adding the microbial inoculum is reduced from 90.38% to 78.67, the degradation rate is 12.9%, OM with adding TB62 microbial inoculum is reduced from 90.67% to 77.63%, and the degradation rate is 14.4% (figure 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 TB62 microbial inoculum is reduced from 71.4mg/g to 9.2mg/g by 87.1% (FIG. 8).

In conclusion, the TB62 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 of Geobacillus thermodenitrificans TB62

The surfactant production condition of the three strains is identified by a blood agar plate and a 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 prove that the geobacillus thermosulfidovorus TB62 can generate the 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> a geobacillus thermosiphonii strain TB62 and application thereof in promoting compost maturity

<130>ZYWS

<160>1

<170>PatentIn version 3.3

<210>1

<211>1458

<212>DNA

<213> Artificial sequence

<400>1

tggggggggt gctatacatg cagtcgagcg gaccgaacga gagcttgctc ttgttcggtc 60

agcggcggac gggtgagtaa cacgtgggca acctgcccgc aagaccggga taactccggg 120

aaaccggagc taataccgga taacaccaaa gaccgcatgg tctttggttg aaaggcggct 180

tcggctgtca cttgcggatg ggcccgcggc gcattagcta gttggtgagg taacggctca 240

ccaaggcgac gatgcgtagc cggcctgaga gggtgaccgg ccacactggg actgagacac 300

ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360

ggagcgacgc cgcgtgagcg aagaaggcct tcgggtcgta aagctctgtt gtgagggacg 420

aaggagcgcc gtttgaataa ggcggcgcgg tgacggtacc tcacgagaaa gccccggcta 480

actacgtgcc agcagccgcg gtaatacgta gggggcgagc gttgtccgga attattgggc 540

gtaaagcgcg cgcaggcggt cctttaagtc tgatgtgaaa gcccacggct caaccgtgga 600

gggtcattgg aaactggggg acttgagtgc aggagaggag agcggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcggctctc tggcctgtaa 720

ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagagg ggtcacaccc tttagtgctg yagctaacgc 840

gataagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960

tcttgacatc ccctgacaac ccaagagatt gggcgttccc ccttcggggg gacagggtga 1020

caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080

agcgcaaccc ttgcctctag ttgccagcat tcagttgggc actctagagg gactgccggc 1140

taaaagtcgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200

cacacgtgct acaatgggcg gtacaaaggg ctgcgaaccc gcgaggggga gcgaatccca 1260

aaaagccgct ctcagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaatcgct 1320

agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380

tcacaccacg agagcttgca acacccgaag tcggtgaggt aacccttacg gagccagccg 1440

ccgaaaggtg ggcaatgt 1458

Claims

1. Geobacillus thermodenitrificans (A)Geobacillus thermodenitrificans) The strain TB62 is preserved in the China general microbiological culture Collection center on the 05 th month 01 in 2017, and the preservation number is CGMCC NO. 13530.

2. Use of Geobacillus thermosyphus strain TB62 according to claim 1 for degrading lignocellulose.

3. Use of the geobacillus thermosyphon strain TB62 according to claim 1 for the preparation of a surfactant.

4. Use of the geobacillus thermosyphon strain TB62 according to claim 1 for the preparation of laccases, lignin peroxidases, manganese peroxidases, cellulases and hemicellulases.

5. The microbial inoculum is characterized in that the seed solution of the geobacillus thermosyphus TB62 strain in claim 1 is inoculated into L B culture solution after 1% (v/v) sterilization in 1L, the culture solution is cultured for 24h in a shaking table at the temperature of 50 ℃ and the rpm of 120, and the culture solution is collected, so that the microbial inoculum is obtained.

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