CN116676212B - A strain of Brevibacillus aydinogluensis PMBT001 and its application - Google Patents

Abstract

The present invention relates to a strain of Brevibacillus aydinogluensis PMBT001 and its application. Its preservation number is GDMCC.No: 63294; the preservation unit is the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbial Culture Collection Center), and the depository address: Xianlie, Guangzhou City, Guangdong Province 5th Floor, Building 59, Courtyard No. 100, Zhong Road, Guangdong Institute of Microbiology, deposited on March 27, 2022. The gene sequence of Brevibacillus aydinogluensis PMBT001 is shown in SEQ ID NO.1. The present invention uses panda feces as the separation source and uses high-temperature separation to screen out a high-temperature bacterium with three enzyme activities for aerobic compost fermentation. Its suitable growth temperature range is 40-70°C and has high cellulase activity and The protease activity can reach a relative enzyme activity of cellulose of 21.7 μmol/mL, a relative enzyme activity of protease of 0.26 μmol/mL, and a relative enzyme activity of lipase of 10.00 μmol/mL at a high temperature of 50°C.

Description

A strain of Brevibacillus aydinogluensis PMBT001 and its application

Technical field

The invention belongs to the field of microbial technology, and specifically relates to a strain of Brevibacillus aydinogluensis PMBT001 and its application.

Background technique

At present, high-temperature aerobic composting treatment is an important way to solve the problem of solid waste. It has the advantages of protecting the environment, saving raw materials and energy, low investment, and low operating costs. Existing research shows that there is a long high-temperature period in the composting process. During the high-temperature stage of composting, most of the parasites, eggs, spores and pathogenic bacteria in the compost are killed. The high-temperature period is the decomposition of organic macromolecules. The main stage is also an important stage to ensure the harmlessness of compost. During this period, microorganisms are extremely susceptible to high temperature factors, their population size and activity will generally decrease, and the rapid decomposition of organic matter will be inhibited. A large number of studies have shown that the temperature of the pile should be controlled between 45°C and 65°C, especially at 55°C and 60°C, which is the optimal temperature for the degradation of macromolecules such as lignocellulose. Studying microorganisms and their activity characteristics within this temperature range is of great significance to the improvement of composting technology. Therefore, the study of high-temperature resistant bacteria has become a hot topic. Inoculating high-temperature bacteria during composting can increase the number of high-temperature organisms and promote the rapid transformation of certain refractory organic matter, thus effectively improving composting efficiency. Studies have shown that adding high-temperature bacteria improves the conversion of organic matter, accelerates the composting process, reduces odorous gas emissions, stabilizes compost products, and improves compost quality.

Agricultural solid waste, mainly crop straw, livestock manure and dead livestock and poultry carcasses, contains a large amount of cellulose, protein and fat substances. Its composition is complex and difficult to be fully utilized or directly used as a carbon source material by most microorganisms. Transformation and utilization. Research shows that when cellulose-degrading bacteria are used in compost, they can effectively increase the temperature of the compost, speed up the composting process, accelerate the degradation of straw, increase the humus content in the compost, and thereby improve the quality of the compost; while the use of protein- and fat-degrading bacteria in compost can It can effectively degrade dead livestock and poultry carcasses and livestock and poultry manure, eliminate compost odor, activate nutrients, thereby effectively killing pathogenic microorganisms and achieving harmless requirements. Therefore, screening high-temperature-resistant and efficient cellulose, protein and fat-degrading bacteria and inoculating them into compost can promote compost maturity and improve the quality of compost products. Research on aerobic composting of high-temperature strains is very active nowadays, but previous research on high-temperature strains mainly focused on their amylase activity and antibiotic production, while research on high-temperature strains with cellulase, protease and lipase degradation capabilities Still very few.

The bacterial culture cycle is short and the growth rate is fast. It can rely on cheap nitrogen and carbon sources as energy sources to produce enzymes. The enzyme expression level is relatively high and relatively easy to obtain. The enzyme has good thermal stability and the prokaryotic expression control system is relatively simple and convenient. Therefore, bacteria have unique advantages in degrading cellulose, protein and fat.

Contents of the invention

The technical problem to be solved by the present invention is to provide a strain of Brevibacillus aydinogluensis PMBT001 and its application. The Brevibacillus aydinogluensis PMBT001 obtained by screening in the present invention is a high-temperature bacterium capable of producing cellulase, protease and lipase. This strain is applied to aerobic In composting, it can accelerate the composting process of manure and increase the nutrient content.

In order to solve the problems existing in the prior art, the technical solutions adopted by the present invention are as follows:

In the first aspect, the present invention provides a strain of Brevibacillus aydinogluensis PMBT001, whose deposit number is GDMCC.No: 63294; the depository unit is the Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbial Culture Collection Center), and the depository address: Guangzhou, Guangdong Province Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province. The date of preservation is March 27, 2022.

Further, the gene sequence of Brevibacillus aydinogluensis PMBT001 is shown in SEQ ID NO.1.

In a second aspect, the present invention provides the application of Brevibacillus aydinogluensis PMBT0011 in preparing microbial inoculants.

In a third aspect, the present invention provides a microbial inoculant containing Brevibacillus aydinogluensis PMBT001 described in the first aspect.

Further, the viable bacterial count of Brevibacillus aydinogluensis PMBT001 in the microbial inoculant is 1.0×10 ⁸ -5.0×10 ⁸ CFU/g.

Preferably, the number of viable bacteria of Brevibacillus aydinogluensis PMBT001 in the microbial inoculant is 1.3×10 ⁸ CFU/g, and the pH value is 6.0-8.0.

In a fourth aspect, the present invention provides a method for preparing the microbial inoculant described in the third aspect, which includes the following steps:

Brevibacillus aydinogluensis PMBT001 was inoculated into the TSB medium, and cultured at 50-60°C, 160-200r/min for 1 day to obtain a seed liquid; the seed liquid was inoculated into the fermentation medium according to an inoculation amount of 5% (v/v) Medium, shake and culture at 50-60℃ 160-200r/min for 1-2 days to obtain the microbial agent;

The components of the fermentation medium are: 7g of protein pulse, 1g of beef extract, 5g of sodium chloride, 10g of glucose, and 1000mL of distilled water.

In the fifth aspect, the present invention provides the use of Brevibacillus aydinogluensis PMBT001 described in the first aspect or the microbial inoculant described in the third aspect in the preparation of microbial fertilizer.

In a sixth aspect, the present invention provides a microbial fertilizer containing Brevibacillus aydinogluensis PMBT001 described in the first aspect or the microbial inoculant described in the third aspect.

In the seventh aspect, the present invention provides the use of Brevibacillus aydinogluensis PMBT001 described in the first aspect or the microbial inoculant described in the third aspect or the microbial fertilizer described in the sixth aspect in the treatment of high-temperature aerobic composting of feces. Applications.

In an eighth aspect, the present invention provides a high-temperature aerobic composting treatment method, which includes the following steps:

S1: Evenly mix fresh manure with urea, wood chips and shavings to form mixed manure. The mixed manure finally contains a mass fraction of 0.05-0.4% urea and 20-30% wood chips and shavings;

S2: Add the microbial inoculant described in the third aspect above at 0.3-1.0% of the volume of the mixed feces, and then use the stacking method to ferment for 10-30 days.

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

The present invention uses panda feces as the isolation source and uses high-temperature separation to screen out a bacterial strain Brevibacillus aydinogluensis PMBT001 with three enzyme activities for aerobic compost fermentation. Its suitable growth temperature range is 40-70°C and it has the ability to produce cellulase. , protease and lipase functions. It has high cellulase activity and protease activity, and can reach cellulose relative enzyme activity of 21.7 μmol/mL, protease relative enzyme activity of 0.26 μmol/mL, and lipase relative enzyme activity of 10.00 μmol/mL at a high temperature of 50°C. By applying the bacterial strain PMBT001 to cow manure composting in Zhaojue County, it has the effect of promoting the improvement of composting efficiency and the utilization of organic waste. The present invention screens high-temperature enzyme-producing strains to provide advantageous strains for the research and development of manure-degrading microbial agents. It is helpful to solve the problems of air pollution, soil pollution and water pollution caused by the composting process, activate nutrients, improve the composting effect, and has good application potential for the treatment of organic waste.

The preservation information of Brevibacillus aydinogluensis PMBT001 of the present invention is as follows:

Deposit number: GDMCC.No: 63294;

Taxonomic name: Brevibacillus aydinogluensis;

Preservation unit: Institute of Microbiology, Guangdong Academy of Sciences (Guangdong Microbial Culture Collection Center);

Address of the preservation unit: Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province;

Storage date: March 27, 2022.

Description of the drawings

Figure 1 shows the colony morphology and bacterial cell micrograph of Brevibacillus aydinogluensis PMBT001 on TSA medium;

Figure 2 shows the phylogenetic tree of Brevibacillus aydinogluensis PMBT001 constructed based on the 16S rRNA gene sequence;

Figure 3 shows the enzyme production function test of Brevibacillus aydinogluensis PMBT001, in which (left) preliminary screening results for cellulose degradation; (middle) preliminary screening results for protein degradation; (right) preliminary screening results for fat degradation;

Figure 4 shows the temperature changes of compost samples;

Figure 5 shows the C/N changes of compost samples;

Figure 6 shows the changes in seed germination rate (GI) of compost samples;

Figure 7 shows the pH changes of compost samples;

Figure 8 shows the total phosphorus of the compost sample;

Figure 9 shows the changes in total potassium of compost samples;

Figure 10 shows the dynamic changes of the bacterial community during the composting process, where A is the PLS-DA analysis chart, B is the species abundance histogram at the phylum level, and C is the species abundance histogram at the genus level.

Detailed ways

The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the present invention.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

In this application:

Tryptone soy peptone agar medium (TSA medium): 15 g tryptone, 5 g soy peptone, 5 g NaCl, 20 g agar, 1000 mL distilled water.

Tryptone casein liquid medium (TSB medium): 17g tryptone, 3g soy peptone, 5g NaCl, 2.5g K ₂ HPO _{4 ,} 2.5g glucose, 20g agar, 1000mL distilled water.

Congo red solid medium: CMC-Na 2.0g, KH ₂ PO ₄ 0.5g, MgSO ₄ ·7H ₂ O 0.25g, (NH ₄ ) ₂ SO ₄ 1.0g, Congo red 0.2g, agar 20g, distilled water 1000mL.

Rescreening medium with CMC-Na as carbon source: CMC-Na 5.0g, KH ₂ PO ₄ 1.0g, NaNO ₃ 3.0g, KCl 0.5g, MgSO ₄ ·7H ₂ O 0.5g, FeCl ₃ ·6H ₂ O 0.01 g, 1000mL of distilled water.

Skim milk solid culture medium: 2.5g yeast extract, 2g peptone, 1.5g acid hydrolyzed casein, 1.5g casein, 40g skim milk, 20g agar, 1g glucose, 1000mL distilled water.

Protease seed medium: 10g glucose, 20g yeast powder, 5g NaCl, 1g K ₂ HPO ₄ , 0.4g MgSO ₄ , pH 7.0, 1000mL distilled water.

Protease basic fermentation medium: 5g glucose, 10g yeast powder, 1g (NH ₄ ) ₂ SO ₄ , 1g CaCl ₂ , 1g NaCl, 0.5g KH ₂ PO ₄ , 0.3g MgSO ₄ , pH 7.0, 1000mL distilled water.

Victoria blue B solid culture medium: 10g albumen, 3g beef extract, 5g sodium chloride, 20g agar, 25mL olive oil, 1000mL distilled water, Victoria blue (4mg/100mL).

Example 1

This embodiment provides a microbial inoculant. The preparation method of the microbial inoculant is as follows:

The Brevibacillus aydinogluensis PMBT001 plate was streaked and inoculated into the TSA medium, and a single colony was picked and inoculated into the TSB medium, and cultured at 50-60°C, 160-200r/min for 1 day to obtain a seed liquid; the seed liquid was prepared according to the Inoculate 5% (v/v) inoculum into the fermentation medium and culture with shaking at 50-60°C and 160-200r/min for 1-2 days to obtain a microbial agent with an effective viable bacterial count of 1.3×10 ⁸ CFU /g, pH value is 6.0-8.0.

The components of the fermentation medium are: 5-10g of protein pulse, 1g of beef extract, 5g of sodium chloride, 10g of glucose, and 1000mL of distilled water.

Example 2

This embodiment provides a high-temperature aerobic composting treatment method, which includes the following steps:

S1: Fresh cow manure from a large national farm in Zhaojue County, Sichuan Province, China, is uniformly mixed with auxiliary materials urea, wood chips and wood shavings to form mixed manure. The mixed manure finally contains a mass fraction of 0.2% urea, 25% wood chips and wood shavings. The mixed manure contains 0.2% urea, 25% wood chips and shavings by mass to help adjust the carbon-nitrogen ratio.

S2: Add the microbial inoculant described in Example 1 at 0.5% of the volume of mixed feces.

S3: The mixed manure is fermented in the manure treatment plant using the stacking method for 14 days. During the entire composting process, the compost is thoroughly mixed twice. When the temperature of the pile reaches above 50°C, the pile is turned for the first time; when the temperature reaches 60°C, the pile is turned for the second time to maintain aerobic conditions and Uniformity and promote material degradation.

Experimental Example 1 Isolation and Purification of High Temperature Bacteria PMBT001

1.1 Isolation of high-temperature bacteria in aerobic composting

In 2021, panda excrement compost (composting temperature ≥ 50°C) was collected at the Dujiangyan Panda Center in Sichuan Province, placed in sterile sampling bags, sealed, stored in an ice box, transported back to the laboratory, and separated into bacteria within 24 hours.

Weigh the fresh compost sample and shake it in sterile water for 1 hour. Use the 10-fold dilution method to prepare a 10 ^-3 -10 ^-6 bacterial suspension. Take 100 μL of each dilution gradient bacterial suspension and spread it on the plate using tryptone casein. Agar medium (TSA) is used to isolate and culture high-temperature bacteria. The culture temperature is 50℃±2℃ and the culture time is 48h. During the period, the growth of the bacterial colonies is observed and recorded; under this culture condition, the colonies grow rapidly and the colonies are selected. Individual colonies were transferred to tryptic soy agar (TSA) and purified using the dilution plate streaking method until a purified strain was obtained. Pick 1-2 single colonies, transfer the purified strain into tryptone soybean liquid medium (TSB), add 50% glycerol, and store in a -80°C refrigerator.

1.2 Strain identification

1.2.1 Strain morphology

Pick 1-2 single colonies, inoculate them on TSA medium, culture them at 50°C for 1 day, and observe the colony morphology of the strains.

1.2.2 Molecular identification of strains

(1) Reagents: sterile double-distilled water, Mix (Tiangen Biochemical Technology Beijing Co., Ltd.).

(2)Primers

8-27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO. 2) and

1492-1523R: 5'-TAC-GACTTAACCCCAATCGC-3' (SEQ ID NO. 3).

(3) Bacterial DNA extraction

Take a few bacterial cells into a sterile 1.5mL Eppendorf tube, add 500 μl of sterile double-distilled water, freeze the centrifuge tube with liquid nitrogen, place it in a 99°C water bath for 5 minutes, take it out, vortex for 30 seconds, and repeat the above operation once. , centrifuge at 12000r/min, use the supernatant as a template, and store it at -20°C; detect by 1% agarose gel electrophoresis.

(4) Bacterial 16S rRNA gene amplification

16S rRNA gene amplification conditions: Gene amplify the extracted bacterial RNA with universal primers 8-27F: and 1492-1523R, pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1.5 minutes, 30 cycle, with a total extension of 20 minutes at 72°C. The PCR product was purified by Shanghai Sangon EZ Spin Column PCR Product Purification Kit UNlQ-1 column PCR product purification kit (SK1142-N) according to the operating instructions. The purified product was sent to Sangon Bioengineering Co., Ltd. for sequencing.

(5)16S rRNA gene sequence analysis and phylogenetic tree construction

The sequences obtained after sequencing were searched for similarity in NCBI using BLAST software, and the 16S rRNA gene sequence of the published strain with the highest similarity was selected as a reference sequence. Multiple sequence alignment analysis was performed using Clustal X software, and MEGA 7.0 software was used to The N-J method was used to construct a phylogenetic tree and determine the taxonomic status of bacteria.

1.3 Experimental results

Bacteria PMBT001 was isolated from panda feces compost. After culturing on TSA medium at 50°C for 1 day, the colonies were flat and round, with smooth surfaces, thin edges, opaque, and basically neat. The colonies were milky white in color and easy to pick. Gram stain is negative, aerobic, the bacteria are rod-shaped, have spores, one end is enlarged, and the size of the bacteria is 2-4nm, see Figure 1.

The strain PMBT001 was subjected to 16S rRNA sequence determination and BLAST homology comparison in the NCBI database. The similarity between this strain and Brevibacillus aydinogluensis reached 100%. Based on experimental results such as morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence homology analysis, PMBT001 was identified as Brevibacillusaydinogluensis, as shown in Figure 2.

The sequence length of Brevibacillus aydinogluensis PMBT001[27F] is 1251bp, and the 16S rRNA gene sequence of Brevibacillus aydinogluensis PMBT001 is shown in SEQ ID NO.1.

Experimental Example 2 Growth temperature and enzyme production function measurement of high-temperature bacteria PMBT001

2.1 Determination of temperature growth range

Take 10 μL of the strain preserved in glycerol and inoculate it on the TSA medium for activation and culture. Place it in the range of 30°C to 80°C and culture it for 48 hours. Observe and record its growth. According to the measurement results, use 5°C as the unit for further accuracy. Until the maximum and minimum growth temperatures are measured.

2.2 Determination of cellulose degradation ability

2.2.1 Preliminary screening of cellulose degradation ability

Pick 3-5 single colonies of activated bacteria, add 3 mL of sterile water, prepare a bacterial suspension, take 50 μL of bacterial liquid and spread it evenly on the TSA medium, place it in a 50°C biochemical incubator for culture, and observe The strains grow uniformly on the surface of the culture medium. Use a 5mm diameter hole punch to punch the uniformly grown strains together with the culture medium to make a bacterial cake and inoculate it on Congo red solid culture medium. Place it in a 50°C biochemical incubator for 3 days. After -5 days, observe whether a transparent hydrolysis circle is produced at the place where the culture medium is inoculated with bacteria, and measure the diameter of the hydrolysis circle, and set up 3 repetitions.

If the strain cannot produce a hydrolysis circle on Congo red medium and the diameter of the hydrolysis circle does not exceed 5 mm, it is deemed to have no cellulose degrading ability and is recorded as -; if the strain produces a hydrolysis circle with a diameter greater than 5 mm, it is deemed to have the ability. The cellulose degrading ability is recorded as +; if the diameter of the hydrolysis circle produced by the strain is greater than 3cm, it is considered to have strong cellulose degrading ability and is recorded as +++.

2.2.2 Carboxymethylcellulase (CMCase) activity determination

The strains that have passed the primary screening are inoculated into the TSB culture medium to prepare a seed bacterial suspension with a bacterial concentration of about 10 ⁸ CFU, and inoculated into the re-screening culture with the carbon source of CMC-Na at an inoculation amount of 10% (v/v). In a basic Erlenmeyer flask, the strains were cultured with constant temperature shaking at 50°C, and the carboxymethyl cellulase (CMCase) activity of the 3, 4, 5, 6, and 7 d strains was measured respectively, and 3 replicates were set.

(1) Drawing of standard curve

Dry anhydrous glucose at 80°C to constant weight to make a 1mg/mL standard glucose solution. Take 6 test tubes and add 0, 0.2, 0.4, 0.6, 0.8, 1.0mL of standard glucose solution respectively. Add distilled water to 2.0mL and add DNS reagent 1.5mL, boiling water bath for 5min, cool and dilute to 25mL, measure the OD value spectrophotometrically at 540nm, and draw a standard curve.

(2) Preparation of crude enzyme solution

The prepared carbon source is CMC-Na double-sieved culture medium, which is divided into 250 mL triangular flasks. Each bottle is 45 mL. Add 5 mL of seed bacterial suspension and place it in a shaker at 28°C for culture. After 7 days of culture, take 1.5 mL of the culture medium. mL of fermentation broth in a centrifuge tube, centrifuge at 10000r/min for 10min to obtain crude enzyme solution.

(3) Enzyme activity measurement

Enzyme activity determination method: Take 0.1 mL of crude enzyme solution and add 1.9 mL of CMC-Na solution with a mass fraction of 1%. Hydrolyze at a constant temperature of 45°C for 20 minutes, add 1.5 mL of DNS chromogenic solution and carry out boiling water bath for 5 minutes, adjust the volume to 25 mL, measure the color at 540 nm, measure the absorbance (OD) value, compare it with the standard glucose curve, and calculate the glucose from the OD value Quantity (m1). Take another 0.1 mL of the crude enzyme solution, add 1.9 mL of water, and add 1.5 mL of DNS chromogenic solution, bathe in boiling water for 5 minutes, adjust the volume to 25 mL, measure the color at 540 nm, and measure the glucose amount (m2) of the crude enzyme solution. Subtract the amount of glucose (m2) from the amount of glucose (m1) to get the actual amount of glucose obtained by the degradation of 1% CMC solution by CMC enzyme. The amount of glucose A calculated from the optical density value is calculated by the formula. Enzyme activity (unit: μmol/mL): enzyme activity = A × 10 × 1000/20.

2.3 Determination of protein degradation ability

2.3.1 Preliminary screening of protein degradation ability

Spread the strain evenly on the TSA culture medium and place it in a 50°C biochemical incubator for culture. Observe that the strain grows evenly on the surface of the culture medium. Use a hole punch with a diameter of 5 mm to punch the uniformly growing strains together with the culture medium. Make a bacterial cake and inoculate it on the skim milk solid medium. Place it in a 50°C biochemical incubator and cultivate it for 2-4 days. Observe whether a transparent hydrolysis circle is produced at the place where the culture medium is in contact with the bacteria. Measure the diameter of the hydrolysis circle and set up 3 repeat.

If the strain cannot produce a hydrolysis circle on the skim milk medium and the diameter of the hydrolysis circle does not exceed 5 mm, it is deemed to have no protein degradation ability, recorded as -; if the diameter of the hydrolysis circle produced by the strain is greater than 5 mm, it is deemed to have protein degradation ability. , recorded as +; if the diameter of the hydrolysis circle produced by the strain is greater than 3cm, it is considered to have strong protein degradation ability, recorded as +++.

2.3.2 Determination of strain protease activity

(1) Drawing of standard curve

Prepare L-tyrosine standard solution and measure immediately after diluting to 100 μg/mL. Take 1 mL of each diluted solution, add 5 mL of 0.4 mol/L sodium carbonate solution and 1 mL of 1 mol/L Folin-phenol reagent solution, shake evenly, develop color in a 40°C water bath for 20 min, use a spectrophotometer at 680 nm, and measure the results respectively. Absorbance, with absorbance A as the ordinate and tyrosine concentration as the abscissa, draw a standard curve and use the regression equation to calculate the amount of tyrosine (μg) when the absorbance is 1, which is the absorbance constant K value. The K value should be Between 95 and 100.

(2) Preparation of crude enzyme solution

Pick 1 loop of the pure culture stored on the slant and inoculate it into the protease seed medium, culture it at 50°C in a shaker at 150rmp for 12 hours, inoculate it into the protease basic fermentation medium at 1% (v/v), and culture it in a 50°C shaker at 150rmp After 48 h, the supernatant was centrifuged for enzyme activity measurement.

(3) Enzyme activity measurement

The enzyme activity of the crude enzyme solution was determined using the Folin-phenol method. Enzyme activity unit definition: At 40°C, 1 μg of tyrosine is produced from hydrolysis of casein substrate per minute, which is defined as one protease activity unit. Put the 2% mass fraction casein solution into a 40°C constant temperature water bath and preheat it for 5 minutes; add 1mL of crude enzyme solution to 1mL of the preheated casein solution, mix well, put it into a 40°C water bath and incubate it for 10 minutes, then add 2mL 0.4 mol/L trichloroacetic acid to terminate the reaction, take 1 mL of the supernatant and add 0.4 mol/L Na ₂ CO ₃ 5 mL, add 1 mL of Folin-phenol reagent to a 40°C water bath to develop color for 20 min, and then measure the absorbance value at 660 nm. The reaction is based on the reaction of adding water. The system is blank.

Calculate enzyme activity: Read the enzyme activity of the final dilution from the standard curve in U/mL. The enzyme activity of the original solution is calculated according to the following formula: protease activity (U/mL) = A × K × 4 ÷ 10 × n.

In the formula: A: OD value of parallel experiments of fermentation stock solution; K: absorbance constant; n: dilution factor of protease solution; 4: total volume of reaction reagents; 10: reaction time 10 minutes; the results are expressed to integers.

2.4 Determination of fat degradation ability

2.4.1 Preliminary screening of fat degradation ability

Victoria blue B is used as an indicator and turns blue when exposed to acid. Lipase-producing bacteria can produce lipase, which can decompose olive oil added to the culture medium and convert it into fatty acids. Victoria blue B turns blue when exposed to acid. Therefore, the ability to produce lipase can be judged based on the size of the discolored circle produced.

Spread the strain evenly on the TSA culture medium and place it in a 50°C biochemical incubator for culture. Observe that the strain grows evenly on the surface of the culture medium. Use a hole punch with a diameter of 5 mm to punch the uniformly growing strains together with the culture medium. Make a bacterial cake and inoculate it on Victoria Blue B solid medium. Place it in a 50°C biochemical incubator and incubate it for 2-4 days. Observe whether blue color is produced at the place where the bacteria are connected to the medium. Measure the diameter of the discoloration circle and set up 3 replicates. .

If the strain cannot produce a discolored circle on Victoria Blue B medium and the diameter of the discolored circle does not exceed 5 mm, it is deemed to have no fat-degrading ability, recorded as -; if the strain produces a discolored circle with a diameter greater than 5 mm, it is deemed to have fat-degrading ability. ability, recorded as +; if the diameter of the discoloration circle produced by the strain is greater than 3cm, it is considered to have strong fat degradation ability, recorded as +++.

2.4.2 Determination of strain lipase activity

Take several 100ml Erlenmeyer flasks, one as a control flask, and the others as measurement flasks. The specific methods are as shown in Table 1.

Table 1 Lipase activity determination method

Titrate with 0.05M standard sodium hydroxide solution until it is slightly red, and record the volume of NaOH solution used in the titration.

Calculation: The specific activity of lipase is defined as one enzyme activity unit based on the amount of enzyme that hydrolyzes fat to produce 1 μmol of fatty acid per minute with 1 g of lipase at pH 7.5 and 40°C.

In the formula: A is the alkali solution consumption of the sample (m1); B is the alkali solution consumption of the control group (m1); N is the concentration of alkali solution, that is, 0.05 μmol; f is the final dilution factor of the crude enzyme solution; t is the action time (min ).

2.5 Experimental results

It has been determined that the temperature growth range of the bacterium Brevibacillus aydinogluensis PMBT001 is 40-70°C, and it has the ability to produce cellulase, protease and lipase. The results are shown in Figure 3 and Table 2. The relative enzyme activity of cellulose is 21.7 μmol/mL. , the relative enzyme activity of protease is 0.26 μmol/mL, and the relative enzyme activity of lipase is 10.00 μmol/mL. The results show that the high-temperature bacterial strain PMBT001 has good high-temperature resistance and enzyme production capabilities, and has the potential for further development and application.

Table 2 Enzyme production ability of strain PMBT001

Experimental Example 3 Application of Bacterial Strain PMBT001 in Cow Manure

3.1 Compost raw materials and processes

Fresh cow dung comes from a large national farm in Zhaojue County, Sichuan Province, China. The fresh manure is evenly mixed with the auxiliary materials urea, sawdust and shavings to form mixed manure. The mixed manure finally contains a mass fraction of 0.2% urea, 25% wood chips and shavings. The mixed manure is randomly divided into 3 groups. The first group does not add any The microbial inoculant was used as the blank control group (CK). The second group was added with 0.5% of the volume of the microbial inoculant prepared in Example 1 (PMBT001 group), and the third group was added with 0.5% of the volume containing the bacterial strain Bacillus.sp Y3. Microbial inoculants (Y3 group). The reference is Zeng Min. Study on the diversity and copper tolerance of Bacillus derived from livestock and poultry manure [J]. Journal of Environmental Science, 2023, 43(2): 485-495. The mixed manure was fermented in the manure treatment plant using the stacking method for 14 days. An automatic thermometer was used to monitor the daily temperature (Tm) changes of the compost manure at 8:00 a.m. and 6:00 p.m., and the compost around the thermometer (three copies) was collected on days 1, 3, 5, 7, 9, 11, 13, and 14 /day), analyze its physical and chemical properties and microbial characteristics. During the entire composting process, the compost is thoroughly mixed twice. When the temperature of the pile reaches above 50℃, the pile is turned for the first time; when the temperature reaches 60℃, the pile is turned for the second time to maintain aerobic conditions and Uniformity.

3.2 Analysis of physical and chemical properties

In accordance with the Chinese Organic Fertilizer Agriculture Industry Standard (NY/T 525-2021), the pH, organic matter (OM), total nitrogen (N), total phosphorus (P ₂ O ₅ ), total potassium (K ₂ O) and Seed germination index (GI) and other physical and chemical properties were analyzed. The pH value of the 1:5 aqueous solution was detected with a pH meter (INESA PHSJ-3F, China). The organic matter content in compost samples was determined using potassium dichromate titration method. 0.25g of air-dried manure was digested in concentrated H ₂ SO ₄ and H ₂ O ₂ , and total N, P ₂ O ₅ and K ₂ O were determined. The total nitrogen was determined using the Kjeldahl method, and the total P ₂ O ₅ was determined using the molybdenum blue colorimetric method. Total K ₂ O was determined using flame photometry. All experiments were measured three times.

3.3 High-throughput sequencing analysis

3.3.1 16S rRNA V3-V4 sequencing process

For 0.25-0.3g compost sample, use fecal genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract DNA, and use an ultra-trace UV spectrophotometer to detect the purity and concentration of DNA; take the genome with qualified quality 30ng of DNA sample and corresponding fusion primer

338F: 5’-ACTCCTACGGGAGGCAGCAG-3’ (SEQ ID NO.4) and

806R: 5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO.5) configure the PCR reaction system, set the PCR reaction parameters for PCR amplification, use Agencourt AMPure XP magnetic beads to purify the PCR amplification product and dissolve it in Elution Buffer, and paste it Add the label to complete the database creation. Use Agilent 2100 Bioanalyzer to detect the fragment range and concentration of the library. Qualified libraries will be sequenced on the HiSeq platform based on the size of the inserted fragment.

3.3.2 High-throughput sequencing information analysis process

The off-machine data is filtered, and the remaining high-quality Clean data is used for later analysis; the reads are spliced into Tags through the overlap relationship between the reads; the Tags are clustered into OTUs and compared with the database and species annotated; based on the OTUs and annotation results Sample species complexity analysis, species difference analysis between groups, correlation analysis and model prediction, etc.

3.4 Results

3.4.1Physical and chemical results

(1) The temperature changes of the compost samples during the composting process are shown in Figure 4. It can be seen from Figure 4 that the temperature of the pile in the experimental group (PMBT001 group) rises more rapidly, and quickly enters the thermophilic phase on the third day after the start of manure composting and remains stable. , compared with CK (sterile group) and experimental group (Y3 group), it reached the thermophilic stage faster and could maintain a longer high temperature period. The results show that in this study, adding PMBT001 bacterial preparation to aerobic compost can accelerate the manure compost into the thermophilic stage and maintain the compost temperature.

(2) The C/N change of the compost sample during the composting process is shown in Figure 5. It can be seen from Figure 5 that organic matter is generally humified or mineralized by microorganisms during the composting process, accelerating the loss of carbon in the form of _CO2 . Therefore, changes in the C/N ratio in compost samples reflect the microbial activity in the pile. As shown in Figure 5, the C/N in the two groups of piles continued to decrease. The CK group, Y3 group and PMBT001 group dropped from 20.90, 22.76 and 20.19 to 17.08, 19.70 and 12.46 respectively, a decrease of 3.82, 3.06 and 7.73 orders of magnitude respectively. . This result is due to the strong mineralization of carbonaceous compounds by microorganisms in the endothermic phase. It is worth noting that the C/N in the PMBT001 group changed more significantly than the CK group within 14 days, indicating that the microbial activity in the PMBT001 group was more active.

(3) The changes in seed germination rate (GI) of compost samples during the composting process are shown in Figure 6. From Figure 6, it can be seen that GI is an indicator that reflects the maturity of compost. The results showed that the GI of the CK group continued to decrease throughout the composting process, from 79.41% on the 1st day to 34.46% on the 14th day (Figure 6), indicating that the accumulation of harmful substances during the composting process inhibited seed germination. Although the GI of the Y3 group increased from the 7th to the 9th day, it showed an overall downward trend. After the composting stage, the GI of the Y3 group dropped from 71.29% to 37.04%. The GI of PMBT001 showed a trend of first decreasing and then increasing, indicating that after the composting stage, harmful substances in the compost pile were removed. On the 14th day, the GI of PMBT001 (98.63) was greater than 80%, indicating that the compost was mature. This result also shows that PMBT001 can accelerate the manure composting process.

(4) The pH changes of the compost samples during the composting process are shown in Figure 7. It can be seen from Figure 7 that the pH value of the CK group is between 7.34-7.67, the pH value of the Y3 group is between 7.52-7.76, and the pH value of the PMBT001 group is between 7.21-7.21. Between 7.42, the pH value of the CK group and Y3 group was slightly higher than that of the PMBT001 group, but the pH changes in the three groups were not significant.

(5) The changes in total phosphorus and total potassium of the compost samples during the composting process are shown in Figures 8 and 9. From Figures 8 and 9, it can be seen that the total nutrients (total phosphorus and total potassium) gradually increase with the composting process. The nutrient content in the compost samples of the PMBT001 group Richer. The total phosphorus and total potassium in the PMBT001 group were significantly higher than those in the CK group and Y3 group on the 14th day.

3.4.2 High-throughput sequencing results

(1) Overall bacterial community changes: High-throughput sequencing methods were used to analyze the dynamic changes of bacterial communities during the composting process. As shown in Figure 10, the CK group, Y3 group and PMBT001 group obtained a total of 2671, 2508 and 2509 OTUs respectively. PLS-DA analysis based on OTU data showed that the microbial community changed greatly during the composting process; the CK group and the bacterial addition group were separated, indicating that bacterial addition had a greater impact on the changes in the bacterial community during the fermentation process.

In the initial stage of composting, the four dominant bacteria in each sample were Proteobacteria, Bacteroidetes, Firmicutes and Spirochaetes. The relative abundance of Proteobacteria decreased and the relative abundance of Firmicutes increased significantly in both groups. The relative abundance of Firmicutes in the PMBT001 group surged to 68.39% on the 7th day of composting and dropped to 36.69% on the 14th day, while the relative abundance of Firmicutes in the CK group increased slowly throughout the composting process (CK: 16.68-42.24%), and the relative abundance of Firmicutes in the Y3 group continued to increase throughout the composting process (Y3: 6.56-54.49%). The relative abundance of Bacteroidetes continued to decrease in the CK group and Y3 group, and first decreased and then increased in the PMBT001 group.

During the composting process, the microbial community changed greatly at the genus level, with the abundance of most genera decreasing. The dominant bacterial genera in the initial stage are Pseudomonas, Acinetobacter, Petrimonas, Sphaerocheta, Bacteroides and Arcobacter. As the composting process proceeds, the relative abundances of Acinetobacter, Petrimonas, Sphaerocheta, Bacteroides and Arcobacter basically decrease.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the patent scope of the present invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims (10)

1. A plantBrevibacillus aydinogluensisPMBT001, having deposit No. GDMCC No:63294; the collection unit is the department of microbiology of the academy of sciences of Guangdong (collection of microbiological bacterial strains of Guangdong), the collection unit address: the Guangdong province, guangzhou City, first, china, no. 100, no. 59, 5, and Guangdong province microbiological institute, with a storage date of 2022, 03 and 27.

2. The method according to claim 1Brevibacillus aydinogluensisThe PMBT001 is characterized in that the 16S rRNA gene sequence is shown in SEQ ID NO. 1.

3. Claim 1 or 2Brevibacillus aydinogluensisApplication of PMBT001 in preparing microbial agent.

4. A microbial agent is characterized in that: comprising the composition according to claim 1 or 2Brevibacillus aydinogluensis PMBT001。

5. The microbial agent of claim 4, wherein: the saidBrevibacillus aydinogluensisThe number of viable bacteria of PMBT001 in the microbial agent is 1.0x10 ⁸ -5.0×10 ⁸ CFU/g。

6. The microbial agent of claim 4, wherein: the saidBrevibacillus aydinogluensis PMBT001 in the microThe number of viable bacteria in the biological bacterial agent is 1.3X10 ⁸ CFU/g, pH value is 6.0-8.0.

7. The method for preparing the microbial agent according to any one of claims 4 to 6, characterized by comprising the steps of:

will beBrevibacillus aydinogluensis PMBT001 is inoculated in a TSB culture medium and cultured for 1 day at 50-60 ℃ and 160-200r/min to obtain seed liquid; inoculating the seed solution into a fermentation culture medium according to an inoculum size of 5% (v/v), and performing shake culture at 50-60 ℃ for 1-2 days at 160-200r/min to obtain a microbial agent;

the components of the fermentation medium are as follows: 5-10g of peptone, 1g of beef extract, 5g of sodium chloride, 10g of glucose and 1000mL of distilled water.

8. Claim 1 or 2Brevibacillus aydinogluensis Use of PMBT001 or a microbial agent according to any one of claims 4 to 5 for the preparation of a microbial fertilizer.

9. A microbial fertilizer, which is characterized in that: comprising the composition according to claim 1 or 2Brevibacillus aydinogluensis PMBT001 or the microbial agent of any one of claims 4-5.

10. Claim 1 or 2Brevibacillus aydinogluensis Use of PMBT001 or a microbial agent according to any one of claims 4 to 5 or a microbial fertilizer according to claim 9 for the high temperature aerobic composting of manure.