CN117363520A - Bacillus subtilis GJ231 and application thereof - Google Patents

Abstract

The invention discloses a Bacillus subtilis GJ231 and its application. The microbiological classification of the Bacillus subtilis GJ231 is Bacillus subtilis GJ231, the Latin literary name is Bacillus subtilis GJ231, the preservation number is CGMCC No. 28101, and the preservation address is Beijing. No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Chaoyang City. The activity of cellulase produced by Bacillus subtilis GJ231 of the present invention is significantly higher than that of other bacteria, and it has strong ability to degrade cellulose. At the same time, the addition of 5% Bacillus subtilis GJ231 in the present invention contributes to the degradation of cellulose in rapeseed straw and the humification of aerobic compost of cow manure, and effectively improves the compost temperature and the seed germination index of the compost product.

Description

Bacillus subtilis GJ231 and its application

Technical field

The invention relates to Bacillus subtilis GJ231 and its application, and belongs to the field of microbial technology.

Background technique

Cellulose is the most abundant natural polymer organic matter on the earth. As the polysaccharide skeleton of plants, it is widely found in agricultural wastes, forestry wastes and municipal organic solid wastes. Cellulose is composed of microfibers arranged in bundles. Each microfiber is composed of hundreds or thousands of D-pyranose rings connected to each other through β-1,4-glycosidic bonds. Therefore, cellulose has a very complex structure. . The crystallinity and heterogeneity of cellulose are the main factors affecting the degradation of cellulose biomass. Its resource processing is difficult and the utilization rate is low, resulting in a waste of resources. Therefore, cellulose conversion and utilization technology has gradually become a research hotspot.

Biological degradation of cellulose is an economical and environmentally friendly process. This method involves the use of microorganisms. The enzymes produced by microorganisms are beneficial to the selective degradation of cellulose biomass. Compared with other methods, it has low cost and less pollution. Etc. Cellulase is the general name for a group of enzymes that degrade cellulose to produce glucose, mainly composed of exo-β-glucanase, endo-β-glucanase and β-glucosidase. During the saccharification process of cellulose, the internal β-1,4-glycosidic bonds are cleaved by the action of endo-β-glucanase, thereby releasing small fiber particles containing free reducing ends and non-reducing ends. The free end of the chain is then acted upon by exo-β-glucanase to release cellobiose, and finally β-glucosidase acts on cellobiose to release glucose as the final product. Most cellulolytic enzymes are derived from microorganisms, and their enzyme activity and stability vary depending on the source.

However, currently there are not many cellulase-producing strains that are suitable for the degradation of cellulose derived from rapeseed straw and aerobic composting of cow manure. Moreover, there are problems such as high enzyme production cost, poor enzyme activity, and small applicable pH range. Therefore, there is an urgent need to find more microbial strains with high and stable enzyme-producing activity and a wider range of effects.

Contents of the invention

The purpose of the present invention is to provide a Bacillus subtilis GJ231, which has a microbiological classification name of Bacillus subtilis GJ231, a Latin name of Bacillus subtilis GJ231, a preservation number of CGMCC No. 28101, and a preservation address of Beichen West, Chaoyang District, Beijing. No. 3, No. 1 Courtyard, Road; the date of preservation is August 4, 2023. The Bacillus subtilis GJ231 was obtained from rotten straw in fields in Gui'an New District, Guiyang City, Guizhou Province through primary screening and re-screening. The bacterium can produce cellulase and degrade cellulose. Observe the colony morphology of the strain. The colony is milky white, with a smooth and opaque surface, and the bacterial cells are in a semi-moist state.

The ITSrDNA sequence of the above-mentioned Bacillus subtilis GJ231 is shown in SEQ ID NO. 1.

The above-mentioned Bacillus subtilis GJ231 was cultured for liquid enzyme production using sodium carboxymethyl cellulose as the carbon source. After culturing for 7 days at 30°C, the crude enzyme liquid produced by it had the highest filter paper enzyme activity and endo-β-glucanase activity. , respectively 16.76±0.25 and 22.16±0.65U/mL.

At the same time, the present invention also provides a method for preparing a Bacillus subtilis GJ231 liquid inoculant. The above-mentioned Bacillus subtilis GJ231 strain is inoculated into an LB liquid culture medium and cultured in a constant temperature oscillator at 30°C and 150 rpm/min for 10 to 12 hours. Finally, a liquid inoculant is obtained.

The components of the above-mentioned LB liquid culture medium are: 5.0g yeast powder, 10.0g tryptone, 10.0g sodium chloride, 1.0L ultrapure water, pH 7.0-7.5.

Another object of the present invention is: an application of the above-mentioned Bacillus subtilis GJ231 in degrading rapeseed straw.

Another object of the present invention is: an application of the above-mentioned Bacillus subtilis GJ231 in aerobic composting of cow manure.

In the above application, when the compost temperature reaches above 55°C on the 7th day, the compost is maintained above 55°C for 6 days; at the end of composting, 5% Bacillus subtilis GJ231 liquid inoculant is added.

Compared with the prior art, the advantage of the present invention is that the activity of cellulase produced by Bacillus subtilis GJ231 of the present invention is significantly higher than that of other bacteria, and it has strong ability to degrade cellulose. At the same time, the present invention adds 5% Bacillus subtilis (Bacillus subtilis) GJ231 to help degrade the cellulose of rapeseed straw and humify the aerobic compost of cow manure, effectively improving the compost temperature and the seed germination index of the compost product.

Picture description:

Figure 1 shows the Congo red staining results measured on sodium carboxymethyl cellulose plate;

Figure 2 is a glucose standard curve;

Figure 3 is a growth curve of Bacillus subtilis GJ231;

Figure 4 is a schematic diagram of the cellulase activity of Bacillus subtilis GJ231:

Figure 5 shows the phylogenetic tree of Bacillus subtilis GJ231 based on the 16S rRNA gene;

Figure 6 is a schematic diagram of the degradation rate of rapeseed straw after treatment with Bacillus subtilis GJ231;

Figure 7 is a schematic diagram of the changes in cellulose content of rapeseed straw after treatment with Bacillus subtilis GJ231;

Figure 8 is a schematic diagram of the temperature changes of Bacillus subtilis GJ231 when applied to cow manure compost;

Figure 9 is a schematic diagram of changes in germination index of Bacillus subtilis (Bacillus subtilis) GJ231 when applied to cow manure compost.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and drawings.

The main media and reagents involved in this application include:

1. Carboxymethylcellulose sodium liquid culture medium: 10.0g sodium carboxymethylcellulose, 2.0g potassium dihydrogen phosphate, 4.0g ammonium sulfate, 0.244g magnesium sulfate, 1.0g peptone, 1.0L ultrapure water, pH 7.0 -7.5.

2. Sodium carboxymethylcellulose solid medium: 10.0g sodium carboxymethylcellulose, 2.0g potassium dihydrogen phosphate, 4.0g ammonium sulfate, 0.244g magnesium sulfate, 1.0g peptone, 20.0g agar, 1.0 ultrapure water L, pH 7.0-7.5.

3. Carboxymethylcellulose sodium enzyme production medium: 20.0g sodium carboxymethylcellulose, 2.0g ammonium sulfate, 0.3g magnesium sulfate, 0.3g sodium chloride, 3.0g peptone, 4.0g potassium dihydrogen phosphate, dissolved In 1L distilled water, pH=7.0-7.5.

4.LB liquid culture medium: 5.0g yeast powder, 10.0g tryptone, 10.0g sodium chloride, 1.0L ultrapure water, pH7.0-7.5.

5.LB solid culture medium: 5.0g yeast powder, 10.0g tryptone, 10.0g sodium chloride, 20.0g agar, 1.0L ultrapure water, pH 7.0-7.5.

6.1g/L Congo red dyeing solution: Weigh 1.0g of Congo red and dissolve it in an appropriate amount of ultrapure water. Add ultrapure water to adjust the volume to 1L.

7.l mol/L sodium chloride solution: Weigh 58.5g sodium chloride, add ultrapure water to dissolve, and adjust the volume to 1L.

8.1mg/mL glucose standard solution: After the glucose is dried at 80°C to a constant weight, accurately weigh 0.10g and dissolve it in an appropriate amount of ultrapure water, and adjust the volume to 100mL.

9. DNS reagent: Weigh 6.3g of 3,5-dinitrosalicylic acid and 21.0g of sodium hydroxide, dissolve them in 500mL of distilled water that has been boiled for 10 minutes and cooled, and add 185.0g of potassium sodium tartrate at 50°C. Melted phenol 5.0mL, sodium sulfite 5.0g, add ultrapure water to make the volume to 1L.

10. Sodium citrate buffer solution: Prepare 0.1mol/L citric acid solution (Liquid A) and 0.1mol/L trisodium citrate solution (Liquid B). Take 205mL of Liquid A and 295mL of Liquid B, mix with ultrapure water Adjust volume to 1L.

11.1% sodium carboxymethylcellulose: Weigh 1.0g of sodium carboxymethylcellulose and dissolve it in ultrapure water. After cooling, adjust the volume to 100ml.

Example 1 Isolation and purification of strains

1. Domestication: Collect rotten straw samples from Gui'an New District, Guiyang City, Guizhou Province. Weigh 10.0g of the rotten straw sample and add it to a 250mL Erlenmeyer flask containing 90mL sodium carboxymethylcellulose liquid culture medium. Culture at 30°C and 150r/min with shaking. 48h.

2. Separation and purification: Separation adopts the method of dilution and coating on plates. After acclimation, take the culture solution and perform gradient dilution with sterile water to a concentration of 10 ^-5 ~ 10 ^-8 . Take 200 μL of each and apply it to carboxymethyl cellulose. On sodium solid medium, invert the culture in a constant temperature incubator at 30°C for 4 days. Observe the colonies growing on the culture medium. According to the color, size, edge regularity, and surface decoration of the colonies, select larger single colonies, culture them on LB solid medium through multiple plate streaks, and purify repeatedly 4- After 6 generations until a single colony appears, the target single strain is screened.

Example 2 Preliminary screening and re-screening of bacterial strains

1. Preliminary screening: The isolated and purified strains were spotted on sodium carboxymethyl cellulose solid culture medium and cultured for 48 hours under constant temperature conditions of 30°C. Prepare an appropriate amount of Congo red staining solution for staining. After staining for 30 minutes, destain with 1 mol/L NaC1 solution for 30 minutes. The staining results are shown in Figure 1. Measure the diameter of the hydrolysis circle and calculate the ratio of the diameter of the hydrolysis circle to the diameter of the colony to initially judge the cellulose degrading ability of the strain. As shown in Table 1, the ratio of the diameter of the hydrolysis circle to the diameter of the colony of GJ231 reaches 2.86.

Table 1 Ratio of hydrolysis circle diameter and colony diameter for Congo red staining of different strains

2. Re-screening: Inoculate the strains with a large ratio of hydrolysis circle diameter to colony diameter obtained from the initial screening into sodium carboxymethylcellulose enzyme-producing medium for cellulase activity measurement.

(1) Drawing of glucose standard curve

Accurately measure 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, 1.4 mL, 1.6 mL and 1.8 mL of glucose standard solutions with a concentration of 1 mg/mL in 25 mL colorimetric tubes. Add pure water to 2 mL, mix the two thoroughly, add 3 mL of DNS reagent to each colorimetric tube, boil in water bath for 5 minutes, cool quickly and adjust the volume to 25 mL with ultrapure water, and place in a 540 nm wavelength UV spectrophotometer. Measure the absorbance of the sample, and draw a standard curve with the glucose content as the abscissa and the absorbance as the ordinate based on the recorded data. The results are shown in Figure 2.

(2) Drawing of strain growth curve

Inoculate the strain into 100 mL of high-temperature sterilized LB liquid culture medium, culture it in a constant-temperature oscillator at 30°C and 150 r/min, take the bacterial liquid every 2 hours, and measure the absorbance at 600 nm with a UV spectrophotometer. Taking the culture time t as the abscissa and the turbidity OD600 of the culture solution as the ordinate, draw the growth curve of the strain to observe its growth cycle and determine the logarithmic growth phase of each bacteria. The results are shown in Figure 3.

(3) Preparation of crude enzyme solution

The strain was cultured in LB liquid medium to the logarithmic growth phase, which was used as the seed liquid for the next step of culturing bacterial enzymes. The bacterial solution was inoculated into 50 mL enzyme-producing fermentation medium at an inoculation amount of 5% (v/v), and cultured continuously at 30°C and 150 r/min for one week. Take 5 mL of fermentation broth every day, centrifuge at 4°C and 10,000 r/min for 10 min, and the supernatant is the crude enzyme solution.

(4) Filter paper enzyme activity assay

Filter paper enzyme activity refers to the total cellulase activity displayed by cellulase consumption of filter paper, which reflects the total effect of the synergistic effect of the cellulase components in the tested sample. The higher the filter paper enzyme activity, the higher the total cellulase activity.

Add 1×6cm filter paper strips to the colorimetric tube, add 1.5mL sodium citrate buffer solution and 0.5mL crude enzyme solution, and use the enzyme solution inactivated by boiling for 10 minutes as a control. After 1 hour in a 50°C water bath, add 3 mL of DNS reagent to each test tube, boil in a boiling water bath for 5 minutes, take it out, immediately cool to room temperature with running water, dilute to 20 mL, and measure the absorbance at a wavelength of 540 nm with a UV spectrophotometer. Find the reducing sugar content on the glucose standard curve and convert the enzyme activity value using the formula. The results are shown in Figure 4. GJ231 had the highest filter paper enzyme activity, reaching 16.76±0.25U/mL on the 5th day.

(5) Endo-β-glucanase activity assay

After obtaining the crude enzyme solution, add 1 mL of 1% carboxymethylcellulose sodium into the colorimetric tubes, and the subsequent experimental steps are the same as (4). The results are shown in Figure 4. The endo-β-glucanase activity of GJ231 was the highest on the 6th day, which was 22.16±0.65U/mL.

(6) Calculation of cellulase activity

The hydrolysis of the substrate to produce 1 μg of glucose in 1 minute by 1 mL of crude enzyme solution is one unit of enzyme activity, expressed in U/mL. The enzyme activity calculation formula is as follows:

X＝(A×N)/(V×T)

In the formula:

X——enzyme activity, U/mL

A——The glucose content obtained on the glucose standard curve according to the absorbance, μgN——The dilution factor of the enzyme solution

V——The amount of enzyme solution added, that is, 0.5mL

T——enzymatic reaction time, i.e. 60min

Example 3 Identification of bacterial species

The genomic DNA of strain GJ231 was extracted, and PCR gene amplification was performed using bacterial 16S rRNA universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). The PCR product was identified by 1% agarose gel electrophoresis, and the PCR product was directly sequenced using PCR primers.

Based on the 16S rRNA sequence, the closest strain at the species level was selected, and MEGA7 software was used to construct a phylogenetic tree of strain GJ231. The results showed that GJ231 is genetically related to various other Bacillus subtilis (Figure 5). The 16SrRNA sequence alignment and phylogenetic tree results further verified that GJ231 is Bacillus subtilis at the molecular biology level.

Example 4 Preparation of Bacillus subtilis GJ231 liquid inoculant

The strain was inoculated into LB liquid culture medium, and cultured in a constant-temperature oscillator at 30°C and 150 r/min for 10-12 hours to reach the logarithmic phase of growth to obtain a liquid Bacillus subtilis GJ231 agent.

Example 5 Degradation effect of Bacillus subtilis GJ231 on cellulose derived from rapeseed straw

Bacillus subtilis GJ231 was applied to the degradation of cellulose derived from rapeseed straw. The rapeseed straw was harvested and naturally air-dried and then crushed to 1-2cm. Weigh 30g of the crushed rapeseed straw sample into a nylon mesh bag and sterilize it at 121°C for 20 minutes before use. The rapeseed straw was soaked with sterile water and evenly mixed with 5% bacterial liquid. It was cultured at 30°C for 14 days, and 5% sterile LB liquid medium was used as a blank control group. After the cultivation, the degradation rate was calculated based on the change in dry weight of rapeseed straw; the change in cellulose content of rapeseed straw before and after degradation was measured.

At the end of degradation, the degradation rates of rapeseed straw in the control group and 5% bacterial solution treatment group were 17.15% and 20.96% respectively. As shown in Figure 6, the cellulose content of rapeseed straw in the control group and 5% bacterial solution treatment group were 54.88% and 54.88% respectively. 51.66%, as shown in Figure 7.

Obviously, the Bacillus subtilis GJ231 obtained in this example can degrade the cellulose component in rapeseed straw, and has the potential to be used in the biodegradation of straw.

Example 6 Effect of Bacillus subtilis GJ231 applied to aerobic composting of cow manure

After collecting cow dung from a breeding farm in Guizhou Province, mix it evenly with 5% bacterial liquid, and conduct an aerobic composting experiment of cow dung in a 60L compost bucket, using 5% sterile culture medium as a blank control group. In order to ensure ventilation, turn the pile every 3 days in the early stage of composting. When the temperature of the compost begins to drop, turn the pile every 7 days. The temperature of the pile was measured and recorded at 10:00 am every day, and compost samples were collected every 7 days for the determination of seed germination index.

The temperature of the cow manure compost in the 5% bacterial agent group was higher than 55°C on the 7th day and remained above 55°C for 6 days. The temperature of the control group reached 55°C on the 18th day and remained there for 1 day, as shown in Figure 8 shown. At the end of composting, the seed germination index of cow manure compost in the 5% inoculant group reached 87%, while the seed germination index of the control group was only 74% (Figure 9).

Obviously, Bacillus subtilis GJ231 can increase the humification rate of cow manure aerobic compost and increase the maturity of cow manure compost products.

Claims (8)

1. A Bacillus subtilis GJ231, characterized by: its microbiological classification name is Bacillus subtilis GJ231, its Latin literary name is Bacillus subtilis GJ231, its preservation number is CGMCC No.28101, and its preservation address is Beichen West Road, Chaoyang District, Beijing No. 3, Courtyard 1. 2. Bacillus subtilis GJ231 according to claim 1, characterized in that: the ITSrDNA sequence of the Bacillus subtilis GJ231 is shown in SEQ ID NO. 1. 3. Bacillus subtilis GJ231 according to claim 1, characterized in that: the Bacillus subtilis GJ231 uses sodium carboxymethylcellulose as a carbon source for liquid enzyme production culture, and after culturing for 7 days at 30°C, The crude enzyme solution produced had the highest filter paper enzyme activity and endo-β-glucanase activity, which were 16.76±0.25 and 22.16±0.65U/mL respectively. 4. A preparation method of Bacillus subtilis GJ231 liquid inoculant, which is characterized in that the Bacillus subtilis GJ231 strain according to claim 1 is inoculated into LB liquid culture medium, and is heated in a constant temperature oscillator at 30° C. and 150 rpm/min. After culturing for 10 to 12 hours, the liquid inoculum is obtained. 5. The preparation method of Bacillus subtilis GJ231 liquid inoculant according to claim 4, characterized in that: the ingredients of the LB liquid culture medium are: yeast powder 5.0g, tryptone 10.0g, sodium chloride 10.0g, Ultrapure water 1.0L, pH 7.0~7.5. 6. Application of Bacillus subtilis GJ231 as claimed in claim 1 in degrading rapeseed straw. 7. Application of Bacillus subtilis GJ231 as claimed in claim 1 in aerobic composting of cow manure. 8. The application of Bacillus subtilis GJ231 in aerobic composting of cow manure according to claim 7, characterized in that: when the compost temperature reaches above 55°C on the 7th day, the compost is maintained above 55°C for 6 days; At the end of composting, add 5% Bacillus subtilis GJ231 liquid inoculant.