Bacillus cereus and application

Abstract

The invention belongs to the technical field of agricultural microorganisms, and relates to bacillus cereus and application thereof. The bacillus cereus is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.33304. The nucleotide sequence of 16S rRNA of the bacillus cereus is as shown in SEQ ID NO: 1. The bacillus cereus can tolerate 100 mM of sodium selenite, has relatively strong selenium salt reducing capacity, and can be used for preparing the selenium-rich bacillus cereus. The method can be applied to selenium pollution treatment and preparation of elemental selenium nanoparticles. In addition, the strain also has IAA secretion activity and capabilities of nitrogen fixation, phosphate solubilization and siderophore production, and can be applied to IAA production, phosphate solubilization, siderophore production and nitrogen fixation. The method is wide in application range and good in market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms and relates to Bacillus cereus and applications. Background Art

[0002] Selenium is an essential trace element for the human body, exhibiting multiple activities within the body. Selenium deficiency can lead to a variety of diseases, including reproductive disorders, immune deficiency, and Keshan disease. However, the human body cannot synthesize selenium internally and must obtain it from the environment. Selenium exists in various oxidized forms in the environment. The oxidized forms of selenium in soil and water primarily include selenate and selenite, both of which are bioaccumulative and highly toxic, causing harm to the human body and serious environmental pollution. Reducing environmental selenium oxides to nanoselenium or selenoamino acids can not only treat selenium pollution but also convert oxidized selenium into forms that are easily absorbed by the human body.

[0003] Microorganisms play a crucial role in the transformation of selenium into its various forms. The search for selenium-reducing bacteria with high tolerance to selenium oxides can not only convert oxidized selenium in the external environment into nano-selenium but also assimilate and reduce selenium oxides in the soil into selenoamino acids. Therefore, the search for selenium-reducing bacteria is an effective means of addressing selenium-induced environmental pollution and providing human-absorbable selenium.

[0004] So far, there have been no reports on selenium-reducing endophytic bacteria. Screening selenium-reducing bacteria from plant endophytic bacteria is of great significance for the control of selenium pollution in the environment and the provision of selenium that can be absorbed by the human body. Summary of the Invention

[0005] Based on the above technical problems, the present invention screens selenium-resistant strains from the endophytic bacteria in the roots of tea trees in selenium-rich tea gardens, identifies and studies the functions of the screened strains, aiming to obtain strains with selenium-reducing effects, and provide more microbial resources for treating selenium pollution in the environment and providing selenium elements that can be absorbed by the human body.

[0006] In one aspect, the present invention provides a Bacillus cereus, the deposit information of the Bacillus cereus is as follows:

[0007] Strain name: GX6-3;

[0008] Classification name: Bacillus cereus;

[0009] Date of receipt by the depository: January 6, 2025;

[0010] Time of issuance of the preservation certificate: January 20, 2025;

[0011] Depository: China General Microbiology Center (CGMCC);

[0012] Deposit number: CGMCC No.33304.

[0013] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] The nucleotide sequence of the 16S rRNA of the Bacillus cereus is shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1 is as follows:

[0015]

[0016] On the other hand, the present invention also provides a bacterial agent, wherein the active ingredient of the bacterial agent is the Bacillus cereus.

[0017] In another aspect, the present invention further provides a bacterial agent for promoting plant growth, wherein the active ingredient of the bacterial agent is the Bacillus cereus.

[0018] Furthermore, in the bacterial agent, the Bacillus cereus has at least one ability of producing IAA, solubilizing phosphate, producing siderophores and fixing nitrogen.

[0019] In addition, the present invention also provides the use of the Bacillus cereus in reducing selenium salts to elemental selenium nanoparticles.

[0020] Furthermore, in the application, the Bacillus cereus reduces sodium selenite into elemental selenium nanoparticles.

[0021] In another aspect, the present invention further provides the use of the Bacillus cereus in the treatment of selenium pollution, wherein the Bacillus cereus reduces selenite to elemental selenium nanoparticles.

[0022] In addition, the present invention also provides the use of the Bacillus cereus in the preparation of elemental selenium, wherein the Bacillus cereus reduces selenite to elemental selenium nanoparticles.

[0023] In addition, the present invention also provides the use of the Bacillus cereus in a selenium-pollution-tolerant environment, characterized in that the Bacillus cereus can grow normally when the concentration of sodium selenite is not higher than 100 mM.

[0024] Finally, the present invention also provides the use of the Bacillus cereus in any one of IAA production, phosphate solubilization, siderophore production and nitrogen fixation.

[0025] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0026] (1) The present invention screened a selenium-resistant bacterial strain with selenium reduction function from endophytic bacteria in the roots of tea trees in a selenium-rich tea garden. The strain is deposited in the General Microbiology Center of the China Culture Collection Administration Committee with a deposit number of CGMCC No. 33304. The strain can grow normally in 100 mM sodium selenite, and the conversion rate of reducing sodium selenite to nano-selenium is as high as 99.57% when the sodium selenite concentration is 1 mM. The output of nano-selenium reaches 2.92 mM when the sodium selenite concentration is 5 mM. The strain has strong selenium reduction ability and strong biological activity for preparing nano-selenium, and can be used in selenium pollution control and in the preparation of nano-selenium.

[0027] (2) By exploring the functions of Bacillus cereus GX6-3, the present invention found that the strain also has the biological activity of secreting IAA, and has the ability to fix nitrogen, solubilize phosphate, and produce siderophores, indicating that it can be used in producing IAA, solubilizing phosphate, producing siderophores or fixing nitrogen.

[0028] The Bacillus cereus GX6-3 provided by the invention has wide biological activities and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram showing the results of sample disinfection treatment.

[0030] Figure 2 Figure 2 is the colony image of endophytic bacteria obtained by screening.

[0031] Figure 3 This is the result of screening for selenium-resistant and selenium-reducing bacteria.

[0032] Figure 4 This is the phylogenetic tree of Bacillus cereus GX6-3.

[0033] Figure 5 The selenium reduction efficiency and nano-selenium production of Bacillus cereus GX6-3 under different selenite concentrations.

[0034] Figure 6 This is the scanning electron microscopy result of Bacillus cereus GX6-3 reducing nano-selenium when 5mM selenite was added to the culture medium.

[0035] Figure 7 This is the result of energy dispersive spectrum analysis (EDX) of the reduction of nano-selenium by Bacillus cereus GX6-3 when 5 mM selenite was added to the culture medium.

[0036] Figure 8 Figure 1 shows the results of a test on the phosphate solubility of Bacillus cereus GX6-3. A shows the results of a test on the ability to solubilize inorganic phosphorus, while B shows the results of a test on the ability to solubilize organic phosphorus.

[0037] Figure 9 This is a graph showing the siderophore production ability test results of Bacillus cereus GX6-3.

[0038] Figure 10 This is a graph showing the nitrogen fixation ability test results of Bacillus cereus GX6-3. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0040] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0041] The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0042] Example 1

[0043] This example is about isolating endophytic bacteria from the roots of tea trees in a selenium-enriched tea garden.

[0044] Sample source: Roots of 'Shaancha No. 1' grown in a selenium-rich tea garden in Ankang City, Shaanxi Province.

[0045] Prepare LB solid medium: weigh 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar powder, dissolve them in distilled water, make up to 1 L with distilled water, and sterilize by autoclaving at 121°C for 21 min.

[0046] Prepare LB liquid medium: weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, dissolve them in distilled water, dilute to 1 L with distilled water, and sterilize under high pressure at 121°C for 21 min.

[0047] Strain isolation and purification: The dilution plate method was used to isolate and purify the endophytic bacteria in the tea tree roots. The collected tea tree roots were washed with clean water, and then the surface moisture was absorbed with filter paper and then the surface was disinfected. The disinfection method is: first place the sample in 75% alcohol, soak, rinse and shake for 3 minutes, then pour out the alcohol, and then immerse in 3% sodium hypochlorite solution for 3 minutes, and finally rinse with sterile water 3 to 4 times. Absorb the last rinse liquid in the surface disinfection process and spread it on the LB plate, and culture it at 28°C for 1 to 3 days. If no microorganisms grow on the culture medium, it proves that the surface disinfection is thorough, and the results are as follows Figure 1 As shown, it shows that the sample is thoroughly disinfected and can be used for subsequent endophyte isolation and purification.

[0048] The above disinfected sample (tea tree root) was added with 100 mL of sterile water and the root was fully ground into a homogenate to prepare a suspension. 1 mL of the suspension was taken and the suspension was divided into 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5For gradient dilution, 100 μL of each gradient dilution solution was spread onto LB solid plates and cultured at 37°C for 2 days.

[0049] A single colony was picked and purified three times using the plate streak method. The results were as follows: Figure 2 As shown, pure culture of the strain was obtained and the strain was selected and stored at 4°C.

[0050] Example 2

[0051] This example is to screen selenium-resistant selenium-reducing strains.

[0052] Preparation of LB culture plates with different sodium selenite concentrations: Weigh 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar powder and dissolve them in distilled water. The volume is made up to 1 L with distilled water and sterilized at 121°C for 21 min to prepare LB solid culture medium. The prepared selenite mother solution is filtered and sterilized, and then added to the LB solid culture medium to prepare sodium selenite media with sodium selenite contents of 1 mM, 10 mM, 50 mM, and 100 mM, respectively. Before cooling, pour the plates to prepare LB culture plates with different sodium selenite concentrations.

[0053] Screening of selenium-tolerant strains: Six strains were randomly selected from the strains isolated and purified in Example 1 and stored at 4°C for streak activation. Single colonies were picked and inoculated into liquid LB medium, shaken at 180 rpm and 28°C for 8 h, and the culture product was used as seed liquid. The seed liquid was added to the liquid LB medium and shaken to make the bacterial liquid OD 600 =0.8, dilute the original bacterial solution to obtain gradient diluted bacterial solution for use. The gradient diluted bacterial solution dilution degrees are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 2.5 μL of the original bacterial solution and the above-mentioned gradient diluted bacterial solution were respectively added to LB culture plates with different sodium selenite concentrations. Three replicates were set for each treatment. After the bacterial droplets were dried, the plates were inverted and cultured at 37°C. After 48 hours of culture, the color, size and number of the colonies were observed to determine the strain's tolerance to selenium. The results are as follows: Figure 3 As shown, the growth of the strain was not affected at a sodium selenite concentration of 100 mM, and the red colonies were identified as highly tolerant selenium-reducing bacteria, which were named GX6-3.

[0054] Example 3

[0055] This example is the identification of strain GX6-3.

[0056] The 16S rRNA gene of the selenium-resistant, reductive bacteria GX6-3 obtained by screening was amplified using bacterial PCR universal primers, and the PCR product was sequenced. The PCR primer sequences are as follows:

[0057] 27F:5'-AGAGTTTGATCCTGGCTCAG-3',

[0058] 1492R:5'-CGGCTACCTTGTTACGACTTC-3'.

[0059] PCR reaction system: 2.5 μL each of 5 μM upstream and downstream primers, 5 μL template DNA, 25 μL 2× Vazyme Lamp MasterMix, and ddH2O to 15 μL.

[0060] PCR reaction program: 94°C for 30 s; 94°C for 15 s, 55°C for 15 s, 72°C for 30 s, 30-35 cycles; 72°C for 2 min.

[0061] The PCR product was sent to a biotechnology company for sequencing, and the sequencing result is shown as SEQ ID NO: 1.

[0062] The sequencing results were BLASTed on the NCBI website, and homologous sequences with high similarity were selected. The neighbor-joining method was used to construct a phylogenetic tree using MEGA11.0 software to determine the species category of the strain. The results showed that the 16S rRNA gene sequence of the GX6-3 strain was most similar to the 16S rRNA gene sequence of the Bacillus cereus NBRC 15305 strain, and they were clustered into one branch. The phylogenetic tree of GX6-3 is shown in Figure 1. Figure 4 As shown, it was shown that the GX6-3 strain was Bacillus cereus, and was named Bacillus cereus GX6-3.

[0063] Example 4

[0064] This example is an analysis of the selenium reducing ability of Bacillus cereus GX6-3.

[0065] A single colony of Bacillus cereus GX6-3 (GX6-3) was selected and inoculated into LB liquid medium at 28°C and 180 rpm until OD 600=0.8, take the above bacterial solution and inoculate it into LB liquid culture medium containing 1mM, 2.5mM, 5mM, 10mM, 15mM, and 20mM sodium selenite at a 0.1% (v / v) inoculation amount, and culture at 28°C and 180rpm for 48 hours.

[0066] Centrifuge the above-mentioned bacterial culture at 10,000 rpm for 8 minutes, remove the supernatant, and rinse three times with distilled water. Add 1M Na2S solution (pre-mixed) at a 1:2 ratio of sample volume to pre-centrifugation bacterial culture volume. Mix thoroughly, react for 1 hour, and centrifuge again at 10,000 rpm for 3 minutes. The supernatant is then measured for absorbance at 500 nm. Each sample is measured in triplicate, with three replicates.

[0067] Accurately weigh 2.5mg, 5mg, 7.5mg, 10mg, and 12.5mg of elemental selenium powder into a test tube. Add 25mL of 1M Na2S solution to each tube and shake to fully dissolve. Use a Na2S solution without selenium powder as a control. Incubate for 1 hour and measure the absorbance at 500nm on a spectrophotometer. Repeat three times for each group. Draw a Se-Na2S standard curve.

[0068] The content of reduced elemental selenium was calculated using the standard curve, and the selenium reduction efficiency and yield were calculated using the formula: Selenium reduction efficiency = amount of elemental selenium after reduction / amount of selenium added to Na2SeO3 × 100%

[0069] The reduction efficiency of sodium selenite and the production of nano-selenium by Bacillus cereus GX6-3 at different sodium selenite concentrations were calculated. Figure 5 As shown, Bacillus cereus GX6-3 can reduce sodium selenite to nano-selenium at a lower concentration. When the concentration of sodium selenite is 1 mM, the reduction efficiency of Bacillus cereus GX6-3 to reduce sodium selenite to nano-selenium is 99.57%, which is the highest reduction efficiency for sodium selenite. When the concentration of sodium selenite is 5 mM, the reduction efficiency of sodium selenite to nano-selenium is 58.41%, and the yield of nano-selenium is the highest, which is 2.92 mM.

[0070] Example 5

[0071] This example is the characterization and analysis of bio-nanoselenium synthesized by Bacillus cereus GX6-3.

[0072] Bacillus cereus GX6-3 (OD 600=0.8) was inoculated at a 0.1% (v / v) inoculum into sterilized LB liquid medium containing 5 mM sodium selenite and cultured at 28°C, 180 rpm, for 48 h. The shake-cultured red bacterial liquid was centrifuged at 10,000 rpm for 8 min at room temperature, the supernatant removed, and the pellet resuspended in physiological saline and rinsed three times by centrifugation to form the sample bacterial liquid.

[0073] A coverslip with a side length of less than 7 mm was placed in the sample solution. After the sample adhered to the coverslip, it was fixed with 2 mL of 5% by volume glutaraldehyde and incubated at 4°C overnight. The sample was then rinsed two to three times with 2 mL of 0.1 M PBS buffer (pH 7.2) for 10 minutes each. The sample was then rinsed once with 30%, 50%, 70%, 80%, and 90% ethanol for 10 minutes each, followed by two 100% ethanol rinses for 10 minutes each. Finally, the sample was replaced with 1 mL of isoamyl acetate for 10 minutes. After drying, the sample was observed and photographed using a field emission scanning electron microscope (FESEM, S-4800), and the nanoparticles were analyzed using an energy dispersive spectrometer (EDX).

[0074] The results are as follows Figure 6 As shown in the figure, a large number of spherical particles were clearly observed outside the Bacillus cereus GX6-3 under the scanning electron microscope. Figure 7 As shown, the Bacillus cereus GX6-3 nanoparticles showed a characteristic absorption peak of selenium at 1.37keV. These results indicate that Bacillus cereus GX6-3 can reduce sodium selenite to produce red elemental selenium nanoparticles.

[0075] Example 6

[0076] This example is a test of the growth-promoting performance of Bacillus cereus GX6-3.

[0077] (1) Test of IAA production capacity of Bacillus cereus GX6-3

[0078] Prepare nitrogen-containing culture medium: weigh 0.5 g yeast extract, 10 g glucose, 1.0 g (NH4)2SO4, 2 g K2HPO4, 0.5 g MgSO4·7H2O, and 0.1 g NaCl and dissolve them in distilled water. Then dilute to 1 L with distilled water and adjust the pH to 7.2-7.4.

[0079] Prepare Salkowski colorimetric reagent: measure 1 mL of 0.5 mol / L FeCl3 and 50 mL of 35% HClO4 and mix them thoroughly.

[0080] Bacillus cereus GX6-3 was inoculated at a 1% inoculum into a nitrogen-containing medium containing 0.2 g / L tryptophan and incubated on a shaker at 28°C and 180 rpm for 48 hours. The culture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was mixed with an equal volume of Salkowski reagent. Positive and negative controls were also set up: the positive control consisted of 800 μL of a 100 mg / L IAA standard solution and an equal volume of Salkowski reagent colorimetric solution, while the negative control consisted of 800 μL of nitrogen-containing medium and an equal volume of Salkowski reagent colorimetric solution. The reaction was allowed to proceed at room temperature in the dark for 30 minutes, and the results were observed. A positive result, which displayed a pink color, indicates that the strain secreted IAA; a darker pink indicates a higher IAA secretion capacity. A negative result, which displayed a color consistent with the negative control (the color of the culture medium), indicates that the strain did not secrete IAA. A pink color indicates that Bacillus cereus GX6-3 is capable of IAA production.

[0081] (2) Phosphate solubilization ability test of Bacillus cereus GX6-3

[0082] Prepare inorganic phosphorus solid culture medium: weigh 0.5 g yeast extract, 10 g glucose, 5 g tricalcium phosphate, 0.5 g (NH4)2SO4, 0.2 g KCl, 0.1 g MgSO4, 0.1 mg MnSO4, 0.1 mg FeSO4, and 15 g Agar, dissolve them in distilled water, and then dilute to 1 L with distilled water.

[0083] Prepare organophosphorus solid culture medium: weigh 10 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of NaCl, 0.3 g of MgSO4, 0.03 g of MnSO4, 0.3 g of K2SO4, 0.03 g of ferrous sulfate, 5 g of calcium phosphate, 0.2 g of lecithin, and 15 g of Agar, dissolve them in distilled water, and then dilute to 1 L with distilled water.

[0084] Bacillus cereus GX6-3 was inoculated into LB liquid medium and cultured until OD 600 =0.8, then the cultured bacterial solution was inoculated onto an inorganic phosphorus solid culture medium and cultured in the dark at 28°C for 7 days. The results were observed. As shown in the figure, a transparent phosphate-dissolving ring appeared around the colony, indicating that the strain had the ability to dissolve phosphate. Figure 8 As shown, Figure 8 A in the middle is the test result of the inorganic phosphorus decomposition ability of Bacillus cereus GX6-3. Figure 8B in the figure is the test result of the ability of Bacillus cereus GX6-3 to degrade organic phosphorus. The results show that Bacillus cereus GX6-3 has the ability to degrade organic phosphorus and inorganic phosphorus, and its ability to degrade organic phosphorus is greater than its ability to degrade inorganic phosphorus.

[0085] (3) Detection of the ability of Bacillus cereus GX6-3 to produce siderophores

[0086] Prepare CAS assay medium: weigh 0.0729 g of HDTMA, 0.0605 g of CAS, 0.3 g of sodium dihydrogen phosphate dihydrate, 1.2135 g of disodium hydrogen phosphate dodecahydrate, 0.125 g of ammonium chloride, 0.0375 g of potassium dihydrogen phosphate, 0.0625 g of sodium chloride, 0.003 g of FeCl3·6H2O, and 9 g of Agar, dissolve them in distilled water, and then dilute to 1 L with distilled water.

[0087] Bacillus cereus GX6-3 was inoculated onto CAS medium and cultured in a 28°C incubator for 7 days. The results were observed. If a yellow halo appeared around the strain, it indicated that the strain had the ability to produce the vector. Figure 9 As shown, the results showed that Bacillus cereus GX6-3 has the ability to produce siderophore.

[0088] (4) Detection of nitrogen fixation ability of Bacillus cereus GX6-3

[0089] Prepare Ashby solid medium: weigh 10 g of mannitol, 0.2 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.2 g of NaCl, 0.1 g of CaSO4·2H2O, 5 g of CaCO3, and 20 g of agar powder, dissolve them in ddH2O, and make up to 1 L with ddH2O. Adjust the pH to 7.4.

[0090] Bacillus cereus GX6-3 was inoculated on Ashby solid medium and cultured at 28℃ for 2 days. The culture was repeated 3 times to observe the growth of the strain. Figure 10 As shown, it was shown that the strain Bacillus cereus GX6-3 could grow normally, indicating that the strain Bacillus cereus GX6-3 had the ability to fix nitrogen.

[0091] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A Bacillus cereus, characterized in that The Bacillus cereus is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 33304.

2. A bacterial agent, characterized in that The active ingredient is the Bacillus cereus according to claim 1.

3. A bacterial agent for promoting plant growth, characterized in that: The active ingredient is the Bacillus cereus according to claim 1.

4. The bacterial agent according to claim 3, wherein the Bacillus cereus has at least one of the abilities of producing IAA, solubilizing phosphate, producing siderophores, and fixing nitrogen.

5. Use of the Bacillus cereus according to claim 1 in reducing selenium salts to elemental selenium nanoparticles.

6. The use according to claim 5, characterized in that The Bacillus cereus reduces sodium selenite to elemental selenium nanoparticles.

7. The use of the Bacillus cereus according to claim 1 in the treatment of selenium pollution, characterized in that: The Bacillus cereus reduces selenite salts to elemental selenium nanoparticles.

8. The use of the Bacillus cereus according to claim 1 in the preparation of elemental selenium, characterized in that: The Bacillus cereus reduces selenite to elemental selenium nanoparticles.

9. The use of the Bacillus cereus according to claim 1 in a selenium-contaminated environment, characterized in that: When the concentration of sodium selenite is not higher than 100 mM, the Bacillus cereus can grow normally.

10. Use of the Bacillus cereus according to claim 1 in any one of IAA production, phosphate solubilization, siderophore production and nitrogen fixation.

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