Geobacillus subterraneus SW2 and application thereof

Abstract

The application discloses a high Bacillus altitudinis SW2, belongs to the field of degrading bacteria screening, and the high Bacillus altitudinis SW2 has been preserved in the China General Microbiological Culture Collection Center on May 8, 2024, has a preservation number of CGMCC No.30525, and has a preservation address of No.3, Xili, Beichen West Road, Chaoyang District, Beijing City; is classified and named as the high Bacillus altitudinis. The strain is obtained through screening, purification and identification technologies from grape branch compost, and through research on strain characteristics, fermentation conditions and cellulase activity of the strain, it is shown that the enzyme activity of the strain is 547.82 U / g under the condition of 30 DEG C and PH=7, and when 5% casein is added as a protective agent, the effective viable cell number of the high Bacillus altitudinis SW2 is the highest and can reach 1.73*10 14 cfu / g.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of degrading bacteria screening and application, and more particularly to a Bacillus altitudinis SW2 and application thereof. BACKGROUND

[0002] Grape is one of the main cultivated fruit trees in China, with a large planting area. According to the data released by the International Organisation of Vine and Wine (OIV), the grape planting area in China in 2020 was about 785,000 hm 2 , and the generated waste grape branches will reach 9.42-11.775 million tons. Composting is a common means of resource utilization of branches. After the branches are crushed and composted, organic fertilizer is made, which is used as base fertilizer. Composting is a physiological and biochemical process dominated by microbial activity, and is also an effective way to realize harmlessness, reduction and resource utilization of grape branches. The composting process is a process of microbial action on organic matter, which can be divided into mineralization and humification. Mineralization is the decomposition of macromolecular organic matter into simple substances, and humification is the generation of more complex humus through microbial activity. After organic waste is composted, it can be used as organic fertilizer in agricultural production, improving soil physical and chemical properties and promoting plant growth.

[0003] However, grape branches contain a large amount of cellulose and lignin, which are difficult to decompose and have a long composting time. The key to the degradation of grape branches lies in the degradation of cellulose and lignin, and microorganisms play an important role in branch composting. In the fermentation of grape branches and other waste, the addition of microbial inoculants can produce cellulase and lignin-degrading enzyme systems, which can promote rapid start of composting, accelerate the process, and significantly improve the quality of organic fertilizer. Therefore, strains play an important role in grape branch fermentation, especially the search for a microbial strain that can efficiently degrade cellulose and lignin.

[0004] Therefore, how to provide a microbial strain that can efficiently degrade cellulose and apply it to branch composting is a problem that those skilled in the art need to solve. SUMMARY

[0005] Therefore, the present application provides a Bacillus altitudinis SW2 and application thereof.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0007] A high altitude bacillus SW2, the strain SW2 has been preserved in the China General Microbiological Culture Collection Center on May 8, 2024, the preservation number is CGMCC No.30525, and the preservation address is No.3, Beichen West Road, Haidian District, Beijing; the classification name is Bacillus altitudinis.

[0008] Preferably, the 16S rDNA sequence of the high altitude bacillus SW2 is shown as SEQ ID No.1.

[0009] As the same inventive concept as the above technical solution, the application also protects the application of the high altitude bacillus SW2 in degrading cellulose.

[0010] Preferably, the application of the high altitude bacillus SW2 in degrading cellulose in branch compost.

[0011] As the same inventive concept as the above technical solution, the application also protects the application of the high altitude bacillus SW2 in producing cellulase.

[0012] As the same inventive concept as the above technical solution, the application also protects a fermentation method of the high altitude bacillus SW2, comprising the following steps:

[0013] (3) the SW2 strain is inoculated on LB medium by streaking, and single bacterial colonies are obtained after constant temperature culture at 30 DEG C;

[0014] (4) the single bacterial colony is inoculated into LB liquid medium, and cultured at 30 DEG C and 180 r / min for 24 h to obtain a culture solution;

[0015] (3) the culture solution is inoculated into sterilized solid medium, and fermented and cultured in a constant temperature incubator at 30 DEG C.

[0016] Preferably, the optimal medium for the strain in step (3) is 5% maltose, 9% casein, 0.5% ammonium chloride and 1.5% potassium dihydrogen phosphate.

[0017] As the same inventive concept as the above technical solution, the application also protects a microbial agent, which comprises the high altitude bacillus SW2 strain.

[0018] Preferably, the agent further comprises a protective agent, and the protective agent is casein.

[0019] As the same inventive concept as the above technical solution, the application also protects the application of the above agent in degrading cellulose.

[0020] Compared with the prior art, the technical effects achieved by the application are:

[0021] The application screens a Bacillus altitudinis SW2 strain from grape branch compost, identifies the species of the strain through 16S rRNA gene sequence analysis, determines the action of the bacteria through cellulase activity, and determines the characteristics of the strain through screening of the optimal temperature, PH, viable cell count and enzyme activity under different transfer times. 10 The enzyme activity is 547.82 U / g at 30 DEG C and PH = 7, the viable cell count can reach 1.67 x 10 14 cfu / mL after 13 transfers, and the enzyme activity is as high as 456.76 U / g after 2 transfers.

[0022] After orthogonal optimization of the fermentation conditions of the Bacillus altitudinis SW2, it is found that the cellulase activity is the highest when the solid culture medium contains 5% maltose, 9% casein, 0.5% ammonium chloride and 0.5% potassium dihydrogen phosphate; and when 5% casein is added as a protective agent, the effective viable cell count of the SW2 strain in the prepared bacterial agent is the highest, reaching 1.73 x 10 14 cfu / g. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of the application.

[0024] The samples and culture media used in the embodiments are as follows:

[0025] Tested sample

[0026] Collected in 2021 from grape branch compost in the hot spring base of the Chinese Academy of Agricultural Sciences.

[0027] Tested culture medium

[0028] LB medium: 10 g of proteose peptone, 5 g of yeast powder, 10 g of sodium chloride, constant volume to 1 L, natural pH, sterilized by high-pressure steam at 121 DEG C for 20 min for standby.

[0029] Cellulose selection medium: 15 g of sodium carboxymethyl cellulose, 5 g of sodium chloride, 1 g of potassium hydrogen phosphate, 0.2 g of magnesium sulfate, 10 g of tryptone, 5 g of yeast powder, 20 g of agar, constant volume to 1 L, pH 7.2, sterilized by high-pressure steam at 121 DEG C for 20 min for standby.

[0030] Rice straw powder solid culture medium: 200g of alkali-treated rice straw powder, 800g of wheat bran, 50g of glucose, 20g of trypsin, 15g of ammonium nitrate, 10g of potassium dihydrogen phosphate, and 1500g of water. Sterilize with high-pressure steam at 121℃ for 20 minutes before use.

[0031] Example 1: Screening and Identification of Bacillus SW2

[0032] 1. Screening of Bacillus SW2 from the Highlands

[0033] (1) Weigh 5g of sample into a sterile 50mL centrifuge tube, add 45mL of sterile water, and shake at 180rpm for 30min. Take 1mL of the mixture and add 9mL of sterile water to perform serial dilutions of the bacterial solution, diluting each solution to 10⁻⁶. -4 10 -5 10 -6 100 μL of the concentrated bacterial suspension was spread onto LB agar plates and incubated upside down in a 30°C incubator. After 3 days, colonies of different morphologies were picked and streaked for purification. The purification method was as follows: colonies were picked up with an inoculation needle and streaked in four non-overlapping areas of an LB agar plate. After colonies grew on the plate, their morphology was observed. If all colonies had the same morphology, the colonies were considered purified.

[0034] (2) The purified strain was inoculated into cellulose selective medium and cultured for 3 days. It was stained with 1 mg / mL Congo red for 1 h, the stain was discarded, and the strain was destained with 1 mol / L NaCl solution. The presence or absence of a transparent zone was observed, and the length and width of the transparent zone were measured. The ratio of the transparent zone area to the colony area was calculated, and the strain with a transparent zone was preserved.

[0035] 2. Identification of Bacillus SW2 from the Highlands

[0036] DNA was extracted from SW2, and its purity and integrity were determined by agarose gel electrophoresis and absorbance measurement. Then, using SW2 DNA as a template, PCR amplification was performed with primers 341F (5'-CGCCCGCCGCG CCCCGCGCCCGGCCCGCCGCCCCCGCCTACGGGAGGCAGCAG-3') and 907R (5'-CCGTCAATTCCTTTGAGTTT-3'). The target band was sent to the company for sequencing, and the obtained DNA sequence was compared with the NCBI database. The results showed that SW2 had 99.83% similarity to Bacillus altitudinis, and was identified as Bacillus altitudinis. The 16S rDNA sequence of this strain is shown in SEQ ID No. 1.

[0037] CGCCCGCCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCGCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAAATTGAC, as shown in SEQ ID NO. 1.

[0038] Example 2 Determination of cellulase activity of Bacillus altitudinis SW2

[0039] The SW2 strain was inoculated into LB liquid medium and cultured at 30°C and 180 r / min for 24 h, and then transferred to rice straw powder solid medium and fermented in a constant temperature incubator at 30°C for 7 d. The cellulase enzyme activity was determined after fermentation according to the method described in “Organic Material Decomposition Agent” (2002).

[0040] The results of enzyme activity determination showed that the cellulase activity of the SW2 strain was 584.47 U / g.

[0041] Example 3 Determination of strain characteristics of Bacillus altitudinis SW2

[0042] Single factor tests were used to investigate the effects of temperature, pH value and transfer times on the cellulase production of the SW2 strain during culture, and the strain characteristics were determined.

[0043] (1) The effect of different temperatures on the cellulase activity of SW2 strain

[0044] The SW2 strain cultured for 24 h was inoculated into the rice straw powder solid medium with natural pH, and was cultured at different temperatures of 20, 30, 40, 50, 60 and 70℃, respectively. The cellulase activity was measured after 7 days. The measurement results are shown in Table 1.

[0045] Table 1 The effect of different temperatures on the cellulase activity of SW2 strain

[0046]

[0047] As shown in Table 1, under different temperature environments, the growth and cellulase activity of the strain are different. The strain SW2 can produce cellulase at 20-70℃, and with the increase of the culture temperature, the cellulase activity shows a trend of first increasing and then decreasing. When the temperature is 30℃, the cellulase activity of the strain SW2 reaches a high level of 330.15 U / g. Therefore, the suitable temperature for the strain SW2 to produce cellulase by fermentation is 30℃.

[0048] (2) The effect of different pH conditions on the cellulase activity of SW2 strain

[0049] The SW2 strain cultured for 24 h was inoculated into the rice straw powder solid medium with pH of 5, 6, 7, 8 and 9, respectively, and was cultured at 30℃. The cellulase activity was measured after 7 days. The measurement results are shown in Table 2.

[0050] Table 2 The effect of different pH conditions on the cellulase activity of SW2 strain

[0051]

[0052] As shown in Table 2, under different pH conditions, the growth and cellulase activity of the strain are different. The strain SW2 can produce cellulase at pH 5-9, and with the increase of the pH, the cellulase activity shows a trend of first increasing and then decreasing. When the pH is 7, the cellulase production capacity of the strain SW2 reaches a high level of 547.82 U / g. Therefore, the suitable pH for the strain SW2 to produce cellulase by fermentation is 7.

[0053] (3) The effect of different transfer times on the effective viable count and cellulase activity of SW2 strain

[0054] The SW2 strain was inoculated into the LB liquid medium, and was cultured at 180 rpm and 30℃. After 24 h of culture, the viable count was measured. The measurement results are shown in Table 3. At the same time, the strain was transferred into new LB medium with an inoculation amount of 1×10 7cfu / mL, the number of transfer was recorded; at the same time of each transfer, the bacteria liquid cultured for 24 hours was inoculated into the rice straw powder solid medium, and cultured in a constant temperature incubator at 30℃ for 7 days, the cellulase activity was measured, and the measurement results are shown in Table 4.

[0055] Table 3 Influence of different transfer times on the effective viable cell number of SW2 strain

[0056]

[0057] Table 4 Influence of different transfer times on the cellulase activity of SW2 strain

[0058]

[0059]

[0060] As shown in Table 3 and Table 4, under different transfer times, the growth of the strain, the effective viable cell number and the cellulase activity are different, and the difference is large; when the transfer time is 14 times, the effective viable cell number of the strain SW2 reaches the highest, which is 1.67x10 10 cfu / mL; when the transfer time is 2 times, the cellulase production capacity of the strain SW2 reaches a high level, which is 456.76U / g.

[0061] Example 4 Optimization of fermentation conditions of Bacillus altitudinis SW2

[0062] The rice straw powder solid medium was used for fermentation condition optimization, and the single factor and orthogonal test were used to investigate the influence of carbon source, nitrogen source (slow-acting nitrogen source and quick-acting nitrogen source) and inorganic salt on the cellulase production of the strain SW2 during the culture, so as to determine the better fermentation conditions.

[0063] 1. Single factor test to determine the types of carbon source, nitrogen source (slow-acting nitrogen source and quick-acting nitrogen source) and inorganic salt

[0064] (1) Carbon source of fermentation medium

[0065] The carbon source was set as fructose, glucose, mannitol, sucrose, lactose, maltose, starch and molasses, etc. 8 treatments, which replaced the glucose in the rice straw powder solid medium; then it was prepared according to the proportion to sterilize the culture medium at 121℃ for 20min, and then inoculated with 1mL of activated SW2 liquid for 18h, and cultured at 30℃, and the cellulase activity was measured after 7d, and the measurement results are shown in Table 5.

[0066] Table 5 Influence of different carbon sources on the cellulase activity of SW2 strain

[0067]

[0068] From Table 5, it can be seen that the addition of different carbon sources in the culture medium will have an impact on the growth of the strain. When the carbon source is maltose, the cellulase activity of the SW2 strain is the highest, reaching 825.75 U / g, so maltose is selected as the optimal carbon source for culturing the SW2 strain.

[0069] (2) Fermentation medium slow-acting nitrogen source and readily available nitrogen source

[0070] The slow-acting nitrogen source was set as casein, yeast powder, trypsin, corn powder, corn syrup, casein, soybean peptone, peanut cake powder, and soybean powder, etc. 9 treatments, replacing the protein peptone in the rice straw powder solid culture medium; then it was prepared into the corresponding culture medium and sterilized at 121°C for 20 min. After the culture medium was cooled, 1 mL of activated SW2 liquid for 18 h was inoculated, and cultured at 30°C. After 7 days, the cellulase activity was measured, and the results are shown in Table 6.

[0071] The readily available nitrogen source was set as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, diammonium hydrogen phosphate, etc. 5 treatments, replacing the ammonium nitrate in the rice straw powder solid culture medium; then it was prepared into the corresponding culture medium, and after sterilization and cooling, 1 mL of activated SW2 liquid for 18 h was inoculated, and cultured at 30°C. After 7 days, the cellulase activity was measured, and the results are shown in Table 7.

[0072] Table 6 Effect of different slow-acting nitrogen sources on cellulase activity of SW2 strain

[0073]

[0074] Table 7 Effect of different readily available nitrogen sources on cellulase activity of SW2 strain

[0075]

[0076] From Tables 6 and 7, it can be seen that when the slow-acting nitrogen source is casein, the cellulase activity of the SW2 strain is the highest, reaching 528.51 U / g, which is significantly higher than that of protein peptone; when the readily available nitrogen source is ammonium chloride, the cellulase activity of the SW2 strain is the highest, reaching 717.64 U / g, which is significantly higher than that of ammonium nitrate. Therefore, casein and ammonium chloride are selected as the optimal slow-acting nitrogen source and readily available nitrogen source for culturing the SW2 strain.

[0077] (3) Fermentation medium inorganic salt

[0078] The inorganic salt was set as calcium carbonate, zinc sulfate, potassium hydrogen phosphate, manganese sulfate, potassium chloride, potassium sulfate, magnesium sulfate, potassium dihydrogen phosphate, sodium chloride, calcium chloride, etc. 10 treatments, replacing the potassium dihydrogen phosphate in the rice straw powder solid culture medium; then it was prepared into the corresponding culture medium, and after sterilization and cooling, 1 mL of activated SW2 liquid for 18 h was inoculated, and cultured at 30°C. After 7 days, the cellulase activity was measured, and the results are shown in Table 8.

[0079] Table 8 Effect of different inorganic salts on cellulase activity of SW2 strain

[0080]

[0081]

[0082] As can be seen from Table 8, when the inorganic salt is potassium dihydrogen phosphate, the cellulase activity of SW2 strain is the highest, up to 548.84 U / g, so potassium dihydrogen phosphate is selected as the optimal inorganic salt for culturing SW2 strain.

[0083] 2. Determination of contents of nutrients in culture medium by orthogonal test

[0084] Based on the orthogonal test design, the contents of maltose, casein, ammonium chloride and potassium dihydrogen phosphate in the solid culture medium of SW2 strain are optimized, and each substance is set at three gradients, as shown in Table 9; SW2 strain is inoculated into the culture medium with corresponding contents for culture, and the cellulase activity is determined after 7 days, and the determination results are shown in Table 10.

[0085] Table 9 Gradient setting of nutrients

[0086]

[0087] Table 10 Results of orthogonal test of nutrient content

[0088]

[0089]

[0090] The orthogonal test results of the nutrient content of SW2 strain are shown in Table 7, and when the solid fermentation conditions of SW2 strain are 5% maltose, 9% casein, 0.5% ammonium chloride and 1.5% potassium dihydrogen phosphate, the cellulase activity of SW2 is the highest, up to 566.39 U / g, which is significantly higher than that of other treatment groups.

[0091] Example 5 Effect of different protective agents on SW2 strain

[0092] SW2 strain is inoculated into rice straw powder solid culture medium, and after 7 days of culture in a constant temperature incubator at 30°C, casein, mannitol, fulvic acid, trehalose and chitosan are selected as protective agents, and the protective agents are added to the solid culture medium containing SW2 strain at a content of 5%, and then the solid culture medium is placed in a 30°C oven for drying until the water content is 5%, and then it is crushed and stored in a sealed bag at room temperature in the dark. After 12 months of storage, the effective viable cell number, the number of miscellaneous bacteria and the cellulase activity are determined according to the standard of "agricultural microbial inoculant" (2006), and the determination results are shown in Table 11.

[0093] Table 11 Influence of different protectants on SW2 bacterial agent

[0094]

[0095] As shown in Table 11, from the effective viable cell number, when 5% casein is used as the protectant, the effective viable cell number of the SW2 strain is the highest, up to 1.73×10 14 cfu / g, which is significantly higher than other components; from the enzyme activity of the bacterial agent, when mannitol and fulvic acid are used as the protectants, the cellulase activity of the prepared bacterial agent is higher, but the effective viable cell number contained is too low. Therefore, the present application selects 5% casein as the protectant, which can not only improve the effective viable cell number of the bacterial agent, but also is beneficial to the preservation of the SW2 strain, and can maintain the cellulase activity of the strain.

[0096] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments, and the same or similar parts between the various embodiments can be referred to each other.

[0097] The above description of disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of highland Bacillus ( Bacillus altitudinis SW2, characterized in that, The strain SW2 was deposited on May 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30525, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; and classified as Bacillus altitudinis.

2. The application of Bacillus SW2 of Highlandiae according to claim 1 in the degradation of cellulose.

3. The application of Bacillus SW2 of Highlandiae according to claim 1 in the production of cellulase.

4. A fermentation method for Bacillus SW2 as described in claim 1, characterized in that, Includes the following steps: (1) Straw SW2 was streaked onto LB medium and cultured at 30°C to obtain single colonies. (2) Inoculate single colonies into LB liquid medium and incubate at 30℃ and 180r / min for 24h to obtain culture solution; (3) Inoculate the culture medium into sterilized solid culture medium and carry out fermentation culture in a constant temperature incubator at 30℃.

5. The fermentation method of Bacillus SW2 according to claim 4, characterized in that, The optimal culture medium for the strain in step (3) is: 5% maltose, 9% casein, 0.5% ammonium chloride and 1.5% potassium dihydrogen phosphate.

6. A microbial inoculant, characterized in that, The bacterial agent includes the Bacillus SW2 strain as described in claim 1.

7. A microbial inoculant according to claim 6, characterized in that, It also includes a protective agent, which is casein.

8. The application of the microbial agent according to any one of claims 6-7 in the degradation treatment of cellulose.

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