Bacillus licheniformis and application thereof

Abstract

The invention discloses bacillus licheniformis and application thereof, and relates to the technical field of microorganisms, the bacillus licheniformis is named as bacillus licheniformis ZY3, the preservation number of the bacillus licheniformis ZY3 is CCTCC NO: M 20241959, the preservation time is September 11, 2024, and the preservation address is China Center for Type Culture Collection. The bacillus licheniformis ZY3 can be used for efficiently reducing exogenous inorganic selenium into nano-selenium, is a non-tryptophan-dependent strain, has high indoleacetic acid secretion capacity and can be used for preparing fertilizers.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Bacillus licheniformis and application thereof. Background Art

[0002] Selenium exists in nature mainly in the form of organic selenium, inorganic selenium and elemental selenium. Compared with the other two forms of selenium, nano-selenium in elemental selenium has the advantages of high activity, low toxicity and easier absorption by the human body, especially nano-selenium synthesized by microorganisms.

[0003] Microbial nano-selenium has a growth-promoting effect on plants. Applying biological nano-selenium to the roots and seedlings of alfalfa can promote the growth of alfalfa and enhance its tolerance to salt stress. Spraying nano-selenium on the leaves of tomato seedlings can increase tomato yield, promote its growth, and improve the nutritional value of tomatoes. Adding biological nano-selenium to the germination stage of rapeseed seeds can promote seed germination and seedling growth and improve photosynthesis capacity.

[0004] However, the selenium tolerance and conversion efficiency of most strains are currently not high. Summary of the invention

[0005] The main purpose of the present invention is to provide a Bacillus licheniformis and application thereof, aiming to solve the problem that most strains in the prior art have poor selenium resistance and poor ability to convert nano-selenium.

[0006] To achieve the above-mentioned object, the present invention proposes a Bacillus licheniformis, which is named Bacillus licheniformis ZY3. The preservation number of Bacillus licheniformis ZY3 is CCTCC NO: M20241959, the preservation time is September 11, 2024, and the preservation address is China Center for Type Culture Collection.

[0007] In one embodiment, the 16S rDNA gene sequence of Bacillus licheniformis is shown as SEQ ID No.1.

[0008] The present invention also provides an application of Bacillus licheniformis in reducing inorganic selenium to nano-selenium. The Bacillus licheniformis includes the aforementioned Bacillus licheniformis.

[0009] The present invention also provides a method for preparing nano-selenium, comprising the following steps:

[0010] The aforementioned Bacillus licheniformis is inoculated into a culture medium containing inorganic selenium, cultured, and extracted to obtain nano-selenium.

[0011] In one embodiment, the inorganic selenium comprises sodium selenite; and / or,

[0012] The concentration of the inorganic selenium is 5-400 mmol / L.

[0013] In one embodiment, the inoculation amount of Bacillus licheniformis in the culture medium is 1%, the concentration of inorganic selenium is 5 mmol / L, the fermentation temperature is 37° C., the fermentation time is 16 h, and the conversion rate of Bacillus licheniformis from inorganic selenium to nano-selenium is 95.5%.

[0014] The present invention also provides an application of Bacillus licheniformis in preparing indoleacetic acid, wherein the Bacillus licheniformis includes the aforementioned Bacillus licheniformis.

[0015] The present invention also provides a method for preparing indoleacetic acid, comprising the following steps:

[0016] The aforementioned Bacillus licheniformis is inoculated into a culture medium, cultured, and separated to obtain indoleacetic acid.

[0017] In one embodiment, the tryptophan concentration in the culture medium is 0 mg / L, the inoculation amount of Bacillus licheniformis is 1%, the fermentation temperature is 37° C., the fermentation time is 48 h, and the amount of indoleacetic acid secreted by the Bacillus licheniformis is 40-50 mg / L; or,

[0018] The tryptophan concentration in the culture medium is 100 mg / L, the inoculation amount of Bacillus licheniformis is 1%, the fermentation temperature is 37° C., the fermentation time is 48 hours, and the amount of indoleacetic acid secreted by the Bacillus licheniformis is 60-70 mg / L.

[0019] The present invention also provides an application of the aforementioned Bacillus licheniformis in agricultural fertilizers.

[0020] In the technical solution of the present invention, the Bacillus licheniformis is named Bacillus licheniformis ZY3, the deposit number of the Bacillus licheniformis ZY3 is CCTCC NO: M20241959, the deposit time is September 11, 2024, and the deposit address is China Center for Type Culture Collection. The Bacillus licheniformis ZY3 can reduce exogenous inorganic selenium to nano-selenium more efficiently, and is a non-tryptophan-dependent strain with a strong indoleacetic acid secretion ability, and can be used to prepare fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0022] Figure 1 This is a colony morphology diagram of the Bacillus licheniformis ZY3 strain in Example 1 provided by the present invention;

[0023] Figure 2 This is a 100-fold Gram staining result of the Bacillus licheniformis ZY3 strain in Example 1 provided by the present invention;

[0024] Figure 3 A phylogenetic tree diagram of the Bacillus licheniformis ZY3 strain in Example 1 provided by the present invention;

[0025] Figure 4 The growth conditions of the Bacillus licheniformis ZY3 strain in Example 2 provided by the present invention on LB solid culture medium containing different concentrations of sodium selenite;

[0026] Figure 5 The growth conditions of the Bacillus licheniformis ZY3 strain in Example 2 provided by the present invention on LB liquid culture medium containing different concentrations of sodium selenite;

[0027] Figure 6 This is a growth curve of Bacillus licheniformis ZY3 in Example 2 provided by the present invention in LB liquid culture medium containing different concentrations of sodium selenite;

[0028] Figure 7 This is a graph showing the nano-selenium production rate of the Bacillus licheniformis ZY3 strain in Example 2 provided by the present invention in the presence of sodium selenite (5 mmol / L);

[0029] Figure 8 This is a scanning electron micrograph of nano-selenium obtained by reducing sodium selenite (5 mmol / L) with the Bacillus licheniformis ZY3 strain in Example 2 provided by the present invention;

[0030] Fig. 9 This is the energy spectrum of nano-selenium obtained by reducing sodium selenite (5 mmol / L) with the Bacillus licheniformis ZY3 strain in Example 2 provided by the present invention;

[0031] Fig.10 This is a particle size distribution diagram of nano-selenium obtained by reducing sodium selenite (5 mmol / L) with the Bacillus licheniformis ZY3 strain in Example 2 provided by the present invention;

[0032] Fig.11 This is a UV-visible spectrum of nano-selenium obtained by reducing sodium selenite (5 mmol / L) with the Bacillus licheniformis ZY3 strain in Example 2 provided by the present invention;

[0033] Fig.12 The nano-selenium obtained by reducing sodium selenite (5 mmol / L) with the Bacillus licheniformis ZY3 strain in Example 2 of the present invention has a wavelength of 400-4000 cm-1 Fourier transform infrared spectrogram in the infrared region;

[0034] Fig.13 This is a graph showing the indoleacetic acid production ability of the Bacillus licheniformis ZY3 strain in Example 3 provided by the present invention.

[0035] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0036] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0037] Selenium exists in nature mainly in the form of organic selenium, inorganic selenium and elemental selenium. Compared with the other two forms of selenium, nano-selenium in elemental selenium has the advantages of high activity, low toxicity and easier absorption by the human body, especially nano-selenium synthesized by microorganisms.

[0038] Microbial nano-selenium has a growth-promoting effect on plants. Applying biological nano-selenium to the roots and seedlings of alfalfa can promote the growth of alfalfa and enhance its tolerance to salt stress. Spraying nano-selenium on the leaves of tomato seedlings can increase tomato yield, promote its growth, and improve the nutritional value of tomatoes. Adding biological nano-selenium to the germination stage of rapeseed seeds can promote seed germination and seedling growth and improve photosynthesis capacity.

[0039] However, the selenium tolerance and conversion efficiency of most strains are currently not high.

[0040] In view of this, the present invention provides a Bacillus licheniformis, which is named Bacillus licheniformis ZY3, the preservation number of Bacillus licheniformis ZY3 is CCTCC NO: M20241959, the preservation time is September 11, 2024, and the preservation address is China Center for Type Culture Collection.

[0041] In the technical solution of the present invention, the Bacillus licheniformis is named Bacillus licheniformis ZY3, the deposit number of the Bacillus licheniformis ZY3 is CCTCC NO: M20241959, the deposit time is September 11, 2024, and the deposit address is China Center for Type Culture Collection, address: Wuhan University, Wuhan, China. The Bacillus licheniformis ZY3 can reduce exogenous inorganic selenium to nano-selenium more efficiently, and is a non-tryptophan-dependent strain with a strong indoleacetic acid secretion ability, and can be used to prepare a plant growth promoter.

[0042] In some embodiments of the present invention, the 16S rDNA gene sequence of Bacillus licheniformis is shown as SEQ ID No.1.

[0043] SEQ ID NO.1:

[0044]

[0045] The present invention also provides an application of Bacillus licheniformis in reducing inorganic selenium to nano-selenium, wherein the Bacillus licheniformis includes the aforementioned Bacillus licheniformis. The Bacillus licheniformis can reduce exogenous inorganic selenium to nano-selenium with high efficiency.

[0046] The present invention also provides a method for preparing nano-selenium, comprising the following steps: inoculating the aforementioned Bacillus licheniformis into a culture medium containing inorganic selenium, culturing, extracting, and obtaining nano-selenium. The culture medium is preferably LB (Luria-Bertani) culture medium.

[0047] In some embodiments of the present invention, the inorganic selenium includes sodium selenite; and / or, the concentration of the inorganic selenium is 5 to 400mmol / L. The concentration of inorganic selenium in the culture medium may be 5mmol / L, 100mmol / L, 200mmol / L, 300mmol / L or 400mmol / L. Preferably, in a solid culture medium, the concentration of the inorganic selenium is 100mmol / L, that is, when the concentration of inorganic selenium is 100mmol / L, the ZY3 strain grows best in a solid culture medium. The culture medium includes a solid culture medium and a liquid culture medium. It should be noted that the ZY3 strain can tolerate a maximum selenium concentration of 400mmoL / L on a solid culture medium and a maximum selenium concentration of 250mmol / L in a liquid culture medium.

[0048] In some embodiments of the present invention, the inoculation amount of Bacillus licheniformis in the culture medium is 1%, the concentration of inorganic selenium is 5mmol / L, the fermentation temperature is 37°C, the fermentation time is 16h, and the conversion rate of Bacillus licheniformis in converting inorganic selenium into nano-selenium is 95.5%. This shows that under the above conditions, the Bacillus licheniformis ZY3 of the present invention has a strong ability to convert inorganic selenium into nano-selenium and a high conversion rate.

[0049] The present invention also provides an application of Bacillus licheniformis in the preparation of indoleacetic acid, wherein the Bacillus licheniformis includes the aforementioned Bacillus licheniformis, and the Bacillus licheniformis can secrete indoleacetic acid in the absence of tryptophan.

[0050] The present invention also provides a method for preparing indoleacetic acid, comprising the following steps: inoculating the aforementioned Bacillus licheniformis into a culture medium, culturing, and separating to obtain indoleacetic acid. The culture medium is preferably LB (Luria-Bertani) culture medium.

[0051] In some embodiments of the present invention, the tryptophan concentration in the culture medium is 0 mg / L, the inoculation amount of Bacillus licheniformis is 1%, the fermentation temperature is 37°C, the fermentation time is 48 hours, and the amount of indoleacetic acid secreted by the Bacillus licheniformis is 40-50 mg / L; or, the tryptophan concentration in the culture medium is 100 mg / L, the inoculation amount of Bacillus licheniformis is 1%, the fermentation temperature is 37°C, the fermentation time is 48 hours, and the amount of indoleacetic acid secreted by the Bacillus licheniformis is 60-70 mg / L. That is, the Bacillus licheniformis ZY3 of the present invention is a non-tryptophan-dependent strain, and the secretion of indoleacetic acid can be increased after adding tryptophan.

[0052] Nano-selenium has a growth-promoting effect on plants, and indoleacetic acid is an important plant hormone that is closely related to plant growth and development. By inoculating the soil with exogenous indoleacetic acid-producing bacteria, the activity of plant antioxidant enzymes can be enhanced to achieve the purpose of promoting crop growth. Therefore, the present invention also provides an application of the aforementioned Bacillus licheniformis in agricultural fertilizers. Bacillus licheniformis has the dual functions of producing nano-selenium and secreting indoleacetic acid, so it is a potential plant growth-promoting bacterium and plays an important role in promoting plant growth, so it can be used as an agricultural fertilizer.

[0053] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0054] Experimental Materials

[0055] The soil was collected from the soil below 10cm near Maotai Town, Guizhou Province.

[0056] LB solid medium: 1% peptone, 1% NaCl, 0.5% yeast powder, 2% agar.

[0057] LB liquid medium: 1% peptone, 1% NaCl, 0.5% yeast powder.

[0058] Example 1 Screening, Isolation and Identification of Bacillus licheniformis ZY3

[0059] 1. Strain Screening

[0060] Soil samples collected from soil below 10 cm near Maotai Town, Guizhou Province were placed in a 65°C oven for 1 hour, and 100 mg of the sample was weighed and diluted with sterile water and spread on LB solid culture medium containing 100, 200, 300, 400, and 500 mmol / L sodium selenite, respectively, and cultured in a 37°C incubator for 48 hours to obtain the cultured strains. The strain with red colony color was selected, purified and named ZY3 strain. The colony morphology of ZY3 strain was observed, and the results are as follows: Figure 1 shown.

[0061] Depend on Figure 1 It can be seen that the colony morphology of the ZY3 strain is a circle with a raised middle and neat edges, a moist surface and a mucous luster; it is short rod-shaped, arranged alone or in pairs side by side, and can be easily connected into long strips.

[0062] 2. Physiological and biochemical identification

[0063] Inoculate ZY3 strain onto LB solid medium, observe and record colony morphology; perform Gram staining and physiological and biochemical identification of the strain according to the Manual of Common Bacterial System Identification. Figure 2 The physiological and biochemical identification results are shown in Table 1.

[0064] Table 1 Physiological and biochemical identification results of strain ZY3

[0065]

[0066]

[0067] Depend on Figure 2 It can be seen that strain ZY3 is a Gram-positive bacterium and conforms to the morphological characteristics of Bacillus licheniformis.

[0068] As shown in Table 1, the ZY3 strain conforms to the experimental results of Bacillus in Bergey's Manual.

[0069] 3. Molecular Biological Identification

[0070] DNA of strain ZY3 was extracted for PCR amplification to obtain PCR amplification products. After the PCR amplification products were confirmed by 1% agarose electrophoresis, they were sent to Shanghai Shenggong Biotechnology Co., Ltd. for 16SrDNA sequencing. The sequencing results were analyzed by Blast homology analysis on NCBI (National Center for Biotechnology Information), indicating that the 16SrDNA similarity between strain ZY3 and model bacteria Bacillus licheniformis strain KP052r was 96%; MEGA7.0 was used to construct a phylogenetic tree (such as Figure 3 Finally, based on the colony and morphological characteristics, strain ZY3 was identified as Bacillus licheniformis.

[0071] Example 2 Identification of the ability of Bacillus licheniformis ZY3 to resist selenium and produce nano-selenium

[0072] 1. Identification of selenium resistance of Bacillus licheniformis ZY3

[0073] (1) Growth of ZY3 strain on LB solid medium containing different concentrations of sodium selenite

[0074] LB solid medium containing 0, 100, 200, 300, 400, and 500 mmol / L sodium selenite was prepared, and single colonies were selected and streaked on the above LB solid medium plates, cultured at 37°C for 48 hours, and the growth of ZY3 strain and the production of nano-selenium on the plates were observed. Figure 4 shown.

[0075] Depend on Figure 4 It can be seen that compared with the LB plate without selenium, the colonies of ZY3 strain in the plate with selenium added are significantly reduced. On the LB plate containing 100mmoL / L sodium selenite, the colonies of strain ZY3 grew well, and red nano-selenium was produced, and the whole colony was red. On the plate containing 400mmoL / L sodium selenite, the growth of the strain and the production of red nano-selenium can still be observed, but the survival rate of the strain is not high. The results show that strain ZY3 can tolerate a maximum selenium concentration of 400mmoL / L on the solid plate.

[0076] (2) Growth of ZY3 strain in LB liquid medium containing different concentrations of sodium selenite

[0077] The ZY3 strain was inoculated into LB liquid medium and cultured at 37°C and 180r / min until the logarithmic phase to prepare the ZY3 strain seed solution. The above strain ZY3 seed solution was inoculated into LB liquid medium containing 0, 50, 100, 150, 200, and 250mmol / L sodium selenite at a 1% inoculation rate, and cultured at 37°C and 180r / min for 48h to observe the production of bacteria and nano-selenium. The results are shown in Figure 5 shown.

[0078] Depend on Figure 5 It can be seen that the culture medium appears dark red to orange because the ZY3 strain reduces sodium selenite to generate nano-selenium, and the LB liquid culture medium containing 100mmoL / L sodium selenite is the darkest red. The results show that the strain ZY3 can tolerate a maximum selenium concentration of 250mmol / L in liquid.

[0079] (3) Growth kinetics of Bacillus licheniformis ZY3 in LB liquid medium containing different concentrations of sodium selenite

[0080] The ZY3 strain seed solution was inoculated into 100 mL LB liquid medium containing different sodium selenite concentrations (0, 5, 25, 50, and 100 mmoL / L) at a concentration of 1%, and cultured at 37°C. The OD was measured every 4 h during the culture process. 600 Values, 3 parallels per group, with culture time as the horizontal axis (h), OD 600 The growth kinetics curve of ZY3 strain was plotted with the value as the ordinate. Figure 6 shown.

[0081] Depend on Figure 6 It can be seen that when the culture medium contains 5, 25, and 50mmol / L sodium selenite, the growth rate of Bacillus licheniformis ZY3 is significantly improved, and it enters the logarithmic growth phase in advance; at this time, the bacterial metabolism is active, resulting in a significant increase in the absorbance value at 600nm, and it enters the logarithmic growth phase in about 3h. Without the addition of selenium, the strain culture reaches the stable period after 24h; while at the concentrations of 5, 25, and 50mmol / L sodium selenite, the time for the ZY3 strain to reach the maximum growth amount is greatly reduced, and the lower the concentration of selenium added, the earlier it reaches the stable period, and the time for the strain to reach the stable period is 16, 20, and 24h, respectively. However, under the condition of 100mmol / L, the growth amount of the strain is low, indicating that this concentration inhibits the growth of the strain. It should be noted that 5mmol / L sodium selenite has a strong promoting effect on the growth of the ZY3 strain and the rate of generating nano-selenium is faster.

[0082] 2. Identification of the ability of Bacillus licheniformis ZY3 to produce nano-selenium

[0083] (1) Nano-selenium production rate of Bacillus licheniformis ZY3

[0084] The ZY3 strain was inoculated into LB liquid medium containing 5 mmol / L sodium selenite and cultured in a shaking incubator at 37°C and 180 r / min for 32 h. Samples were taken every 4 h during the culture process. The samples were centrifuged at 12000 r / min for 30 min to obtain the supernatant. The concentration of residual selenite in the supernatant was quantitatively analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The results are shown in Figure 2. Figure 7 shown.

[0085] After the ZY3 strain had grown for 3 hours, the fermentation liquid began to turn red. Figure 7 It can be seen that after 12 hours of growth of the ZY3 strain, 73.3% of the sodium selenite was reduced to nano-selenium, and after 16 hours, 95.5% of the sodium selenite was reduced to nano-selenium, which greatly shortened the time for the strain to produce nano-selenium. This shows that the ZY3 strain can efficiently reduce sodium selenite and synthesize nano-selenium with high efficiency and short time, which is conducive to shortening the time of industrial production.

[0086] (2) Characterization of Nano-Selenium Reduced by Bacillus licheniformis ZY3

[0087] 1% ZY3 strain seed liquid was added to LB medium containing 5mmol / L sodium selenite, and cultured at 37℃, 180r / min for 48h to obtain culture liquid. The culture liquid was centrifuged at 12000r / min for 15min, and the precipitate was collected; sterile water was added for washing and centrifugation was performed; the precipitate was collected in a mortar and ground with liquid nitrogen. The ground precipitate was dispersed by ultrasound for 6min, and then resuspended with sterile water to obtain a suspension. The suspension was filtered through 20, 10, 5, 3, 1.2, and 0.8μm filter membranes in sequence to remove bacteria, and then the suspension was extracted with 1 / 5 volume of n-hexane, and the extract was centrifuged at 12000r / min for 10min, and the red nano-selenium precipitate was collected and sealed and stored at 4℃.

[0088] The purified nano-selenium was sent to e-Test Technology Co., Ltd. for scanning electron microscopy (SEM) to observe the morphology of nano-selenium. The results are as follows: Figure 8 As shown; and the energy dispersive X-ray spectrum (EDS, Energy-Dispersive X-ray Spectroscopy) of nano-selenium was measured to analyze its chemical composition. The results are as follows Fig. 9 The particle size of nano-selenium synthesized by ZY3 strain was measured by Malvern particle size analyzer. The results are shown in Fig.10 The purified nano-selenium was subjected to UV-visible spectroscopy, and the results are shown in Fig.11 The purified nano-selenium and potassium bromide were placed in a mortar and ground into fine powder, and the powder was immediately placed in an infrared dryer for drying. The spectrum was scanned using a Fourier transform infrared spectrometer, and the test results were analyzed by groups. The results are shown in Fig.12 shown.

[0089] Depend on Figure 8 It can be seen that nano-selenium is spherical, uniform in shape and has good dispersibility.

[0090] Depend on Fig. 9 It can be seen that the characteristic peak of Se (selenium) was detected in the energy spectrum analysis of the red precipitate, confirming the existence of nano-selenium.

[0091] Depend on Fig.10 It can be seen that the average particle size of nano-selenium is 158.8±1.82nm, and the size of nano-selenium particles is an important factor in determining its chemical properties and biological activity. The results show that the ZY3 strain can produce small-sized and highly stable red nano-selenium, enriching the selenium-rich microbial resources.

[0092] Depend on Fig.11 It can be seen that the UV-visible spectrum of the purified nano-selenium shows a peak at 280 nm, which is usually attributed to aromatic amino acids, indicating the presence of protein substances adhering to the surface of the nano-selenium.

[0093] Depend on Fig.12 It can be seen that the purified biogenic nano-selenium has a peak at 3429 cm -1 The absorption peak at 2921cm -1 The absorption peak at 1655 cm is the aliphatic saturated acid and CH stretching vibration of the protein side chain. -1 The absorption peak at 1539cm indicates the presence of CO bonds in different protein structures. -1 The absorption peak at 1458 cm indicates that the peak centered at this point is the stretching of protein amide C=O. -1 1384cm -1 The absorption peak at 1240cm indicates the bending of the CH bond and the existence of CO or CH bond. -1 1063cm -1 The absorption peaks at 900-1200 cm-1 are likely to be typical stretching vibrations of amide III / nucleic acid phosphate and COC groups, which may indicate the presence of carbohydrates and proteins. -1 and 1500-1600cm -1 The absorption peak at 40° indicates the presence of extracellular polysaccharides and proteins, which are the metabolites of bacteria. In general, nano-selenium has characteristic peaks such as amide and carboxyl. It is speculated that nano-selenium contains protein substances. The surface of microbial nano-selenium is coated with a layer of protein, which makes its particle structure stable and evenly dispersed. It can be used as a natural dispersant, thus having good biological activity. Example 3 Identification of the ability of Bacillus licheniformis ZY3 to produce indoleacetic acid

[0094] (1) A single colony of Bacillus licheniformis ZY3 was inoculated into LB liquid medium containing 100 mg / L tryptophan, and cultured at 37°C and 180 r / min for 1 day. After centrifugation at 8000 r / min for 10 min, 50 μL of supernatant was taken and placed in a 2 mL EP tube. 50 μL of Salkowski colorimetric agent was added and mixed evenly. 50 mL of 20 mg / L indoleacetic acid standard solution was used as a positive control. After dark reaction at room temperature for 30 min, the color change was observed. The results showed that the mixed solution of Bacillus licheniformis ZY3 turned pink, indicating that the Bacillus licheniformis ZY3 strain had the ability to produce indoleacetic acid.

[0095] (2) Prepare 10, 20, 30, 40, and 50 mg / L indoleacetic acid standard solutions in sequence, mix with the color developer and measure OD 530 The standard curve of indoleacetic acid was drawn based on the absorbance values ​​at .

[0096] The ZY3 strain seed liquid was inoculated into LB liquid medium containing 0, 100, 200, 300, 400, 500, and 600 mg / L tryptophan, respectively, and cultured at 37°C and 180 r / min for 2 days. The bacterial liquid was then centrifuged at 8000 r / min for 10 min to obtain the supernatant. 1 mL of the supernatant was taken and 1 mL of Salkowski color developer was added to mix. After dark reaction at room temperature for 30 min, the OD was measured. 530 The absorbance value at . The indoleacetic acid content in the bacterial solution was calculated based on the indoleacetic acid standard curve. The results are shown in Fig.13 shown.

[0097] Depend on Fig.13 It can be seen that the ZY3 strain has the ability to produce indoleacetic acid under normal fermentation conditions (i.e., without adding tryptophan, CK group), and the content of indoleacetic acid increases with fermentation time, reaching 47.15 mg / L after 48 hours. After adding 100 mg / L tryptophan, the ability of the ZY3 strain to produce indoleacetic acid increased significantly, reaching 67.41 mg / L after 48 hours, which is 1.43 times that under normal fermentation conditions. This shows that the ZY3 strain can produce indoleacetic acid through a non-tryptophan-dependent pathway, and can also produce indoleacetic acid through the tryptophan pathway in the presence of tryptophan.

[0098] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A Bacillus licheniformis, characterized in that The Bacillus licheniformis is named Bacillus licheniformis ZY3, the preservation number of the Bacillus licheniformis ZY3 is CCTCCNO: M 20241959, the preservation time is September 11, 2024, and the preservation address is China Center for Type Culture Collection.

2. The Bacillus licheniformis according to claim 1, characterized in that The 16S rDNA gene sequence of the Bacillus licheniformis is shown as SEQ ID No.

1.

3. An application of Bacillus licheniformis in reducing inorganic selenium to nano-selenium, characterized in that: The Bacillus licheniformis includes the Bacillus licheniformis according to claim 1 or 2.

4. A method for preparing nano-selenium, characterized in that: The following steps are involved: The Bacillus licheniformis as claimed in claim 1 or 2 is inoculated into a culture medium containing inorganic selenium, cultured, and extracted to obtain nano-selenium.

5. The method for preparing nano-selenium according to claim 4, characterized in that: The inorganic selenium includes sodium selenite; and / or, The concentration of the inorganic selenium is 5-400 mmol / L.

6. The method for preparing nano-selenium according to claim 4, characterized in that: The inoculation amount of Bacillus licheniformis in the culture medium is 1%, the concentration of inorganic selenium is 5 mmol / L, the fermentation temperature is 37° C., the fermentation time is 16 hours, and the conversion rate of Bacillus licheniformis from inorganic selenium to nano-selenium is 95.5%.

7. Use of Bacillus licheniformis in the preparation of indoleacetic acid, characterized in that: The Bacillus licheniformis includes the Bacillus licheniformis according to claim 1 or 2.

8. A method for preparing indoleacetic acid, characterized in that: The following steps are involved: The Bacillus licheniformis as claimed in claim 1 or 2 is inoculated into a culture medium, cultured, and separated to obtain indoleacetic acid.

9. The method for preparing indoleacetic acid according to claim 8, wherein The tryptophan concentration in the culture medium is 0 mg / L, the inoculation amount of Bacillus licheniformis is 1%, the fermentation temperature is 37° C., the fermentation time is 48 hours, and the amount of indoleacetic acid secreted by the Bacillus licheniformis is 40-50 mg / L; or, The tryptophan concentration in the culture medium is 100 mg / L, the inoculation amount of Bacillus licheniformis is 1%, the fermentation temperature is 37° C., the fermentation time is 48 hours, and the amount of indoleacetic acid secreted by the Bacillus licheniformis is 60-70 mg / L.

10. Use of the Bacillus licheniformis according to claim 1 or 2 in agricultural fertilizers.

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