Bacillus megaterium and application thereof in increasing selenium content of tea leaves and preparing organic fertilizer

Abstract

The invention provides bacillus megaterium and application thereof in preparation of a nano-selenium organic fertilizer and improvement of the selenium content of tea leaves, and belongs to the technical field of microorganisms, the bacillus megaterium NT2 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M 20251806, and the bacillus megaterium NT2 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M 20251806. The bacillus megatherium NT2 has relatively strong resistance to reduction on selenium element, can survive in high-concentration inorganic selenium, can reduce the inorganic selenium into nano-selenium, and has relatively weak capability of enriching arsenic, cadmium and lead; the selenium source of the nano-selenium organic fertilizer can be prepared by mixed fermentation with high-selenium rock powder which is calcined and activated at high temperature, so that the content of heavy metals does not exceed the national standard while the selenium content of the tea leaves is increased. The bacillus megaterium provided by the invention not only can reduce the toxicity of inorganic selenium in high-selenium rock and convert the inorganic selenium into nano-selenium, but also can stably, safely and standardly produce selenium-rich tea leaves, the heavy metal content of the tea leaves does not exceed the standard, and the bacillus megaterium has a wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus megaterium and its application in increasing the selenium content of tea and preparing organic fertilizer. Background Technology

[0002] Arsenic, cadmium, and lead are the main heavy metal pollutants in soil, posing significant threats to soil ecology, crop growth, and human health. Heavy metals inhibit the activity of soil microorganisms (such as nitrifying and ammonifying bacteria), affect nitrogen cycling, and reduce soil fertility. Cadmium and lead disrupt the stability of soil aggregates, alter soil pH, and lead to nutrient imbalances. Cadmium and arsenic accumulate in crops; excessive levels inhibit crop growth and can even reduce yields. Cadmium and lead easily accumulate in crops such as rice and vegetables, exceeding food safety standards and threatening food safety. Heavy metals also have significant toxic effects on human health.

[0003] Bacillus megaterium ( Bacillus megaterium Bacillus is a Gram-positive, aerobic, sporulating rod-shaped bacterium belonging to the genus Bacillus. It is widely distributed in soil and has significant agricultural and environmental applications. Its cells are rod-shaped with rounded ends, arranged singly or in short chains, measuring 1.2–1.5 × 2.0–4.0 μm; spores are elliptical, measuring 1.0–1.2 × 1.5–2.0 μm, and are mesophyllary or subterminal. It can secrete organic acids and phosphatases, improving the rhizosphere microenvironment, enhancing root vitality, promoting the absorption and utilization of soil elements by plants, and reducing inorganic selenium to nano-selenium, thus improving the bioavailability and safety of environmental selenium.

[0004] Bacillus megaterium uses functional groups such as carboxyl and phosphate groups on its cell wall surface to complex, exchange ions, or electrostatically adsorb metal ions, fixing heavy metals on the cell surface or inside the cell and reducing the bioavailability of metal ions in the environment. Simultaneously, it secretes specific enzymes to reduce high-valence metal ions to low-valence metal ions, altering their oxidation state and reducing their toxicity. However, Bacillus megaterium generally reduces metal ions indiscriminately; that is, after synthesizing organoselenium (selenomethionine) or nano-selenium, it also adsorbs heavy metal ions such as arsenic, cadmium, and lead into the strain, increasing heavy metal accumulation. If these heavy metal ions are converted into organic heavy metals, their toxicity and absorption rate increase significantly, amplifying the harm of heavy metal pollution.

[0005] Selenium-rich tea refers to tea products with selenium content significantly higher than ordinary tea, mainly produced in areas with high soil selenium content (such as Ankang, Shaanxi, and Enshi, Hubei), and the national agricultural industry standard (NY / T 600-2002) for selenium-rich tea stipulates that the selenium content of selenium-rich tea is 0.25-4.00 mg / kg. According to the results of the inventors' previous research, as one of the main selenium-rich areas in China, the proportion of natural selenium-rich tea in Ankang is less than 26%, and the selenium content of most tea is less than 0.25 mg / kg, the main reason being the point-like distribution and uneven distribution of soil selenium content, leading to unstable tea selenium content. Therefore, using soil microbial inoculants to promote plant absorption of soil selenium or using microorganisms to convert and prepare high-bioavailability selenium fertilizer is a selectable way. SUMMARY

[0006] The purpose of the present application is to improve and stabilize the selenium content in tea without increasing the heavy metal content in tea. A strain of Bacillus megaterium NT2 is selected, which can reduce and enrich inorganic selenium as nano selenium while having low reduction and enrichment ability for arsenic, cadmium, and lead, and nano selenium organic fertilizer is prepared using the nano selenium to root apply tea trees, thereby ensuring that the selenium content of tea is stable and meets the selenium-rich tea standard, and the heavy metal content of tea is not increased.

[0007] The present application first selects a strain of Bacillus megaterium NT2, which was deposited at the China Center for Type Culture Collection on August 8, 2025, with the Latin name: Priestia megaterium NT2, the strain deposit number is CCTCC NO: M20251806, the address is Wuhan University, Wuhan, Hubei Province, China, the postcode is 430072, and the telephone number is 027-68754052.

[0008] The colony of the deposited Bacillus megaterium NT2 is round, as shown in Figure 1 , the color on the LB medium is light yellowish white, the mycelium edge is neat, and the surface is slightly shiny; the color on the selenium-containing medium is red, as shown in Figure 2 .

[0009] The 16S rRNA sequence information of Bacillus megaterium NT2 is as follows:

[0010] Based on the strong selenium-reducing ability of Bacillus megaterium NT2 preserved in this invention, inorganic selenium can be reduced to nano-selenium. Nano-selenium has higher bioavailability and lower toxicity than inorganic selenium. A mixture of nano-selenium and bacterial cells can be prepared by reducing inorganic selenium with Bacillus megaterium NT2, thereby reducing the toxicity of inorganic selenium and improving its bioavailability. Furthermore, nano-selenium organic fertilizer was prepared using the nano-selenium and bacterial cell mixture as a selenium source and applied to the roots of tea trees, increasing the selenium content in tea leaves without significantly changing the heavy metal content compared to the control.

[0011] Therefore, this Bacillus megaterium NT2 can be used to prepare nano-selenium organic fertilizer that increases the selenium content of tea.

[0012] This invention also provides a method for preparing a mixture of nano-selenium and bacterial cells, comprising the following steps: ① Activation of high-selenium rocks: Mix high-selenium rock powder with CaCO3 and calcine at 400~600℃ for 3~5 hours to obtain activated high-selenium rock powder; ② Preparation of high-selenium rock culture medium: After activating high-selenium rock powder is passed through 400 mesh, it is added to LB liquid culture medium, shaken, and then allowed to stand until the high-selenium rock powder settles naturally to obtain high-selenium rock culture medium; ③ Culture of the strain: The cultured Bacillus megaterium NT2 fermentation broth was inoculated into the prepared high-selenium rock culture medium and aerobic fermentation was carried out to obtain selenium conversion fermentation broth; the preservation number of Bacillus megaterium NT2 is: CCTCC NO: M20251806; ④ Collection of nano-selenium and bacterial cell mixture: Centrifuge the fermentation broth to collect the precipitate, then add ddH2O to resuspend the bacterial cells, centrifuge the bacterial cells a second time to collect the red precipitate, wash the red precipitate with ddH2O and freeze-dry to obtain nano-selenium and bacterial cell mixture powder.

[0013] The mass ratio of the high-selenium rock powder to CaCO3 is 1:0.4.

[0014] The aerobic fermentation is specifically carried out at 37±2℃ and 100~300 rpm for 24~48 hours.

[0015] The activated high-selenium rock powder and LB liquid culture medium were in a mass-to-volume ratio of 1:4.

[0016] Simultaneously, using a mixture of nano-selenium and microbial cells as the selenium source, this invention also provides a method for preparing nano-selenium organic fertilizer, comprising the following steps: ① Crush soybean cake, rapeseed cake and dried straw auxiliary materials, mix them evenly with animal manure, and control the carbon-nitrogen ratio to be 25:1~30:1 to obtain an organic matter mixture; ② Spray the fermentation broth of Bacillus megaterium NT2 evenly onto the organic matter mixture at a mass ratio of 1~5%, and adjust the water content of the organic matter mixture to 50%~60% with water. The preservation number of Bacillus megaterium NT2 is: CCTCC NO: M20251806. ③Pile the material in a warm, moist, and aerobic environment with a temperature of 28~37℃ and a humidity of 60~80%; ④ When turning the fermentation pile, add 1~1.5% of a mixture of nano-selenium and microbial cells evenly to the fermentation pile as a selenium source and continue fermentation; ⑤ After fermentation and decomposition, the organic fertilizer components and selenium content are tested and found to meet the standards. Once these conditions are met, fermentation is stopped to obtain nano-selenium organic fertilizer. Beneficial effects

[0017] The inventors investigated the selenium content of tea leaves in Ziyang County, Ankang City, Shaanxi Province, delineating production areas for naturally selenium-rich tea (selenium content ≥ 0.25 mg / kg). Total selenium and available selenium were measured in the rhizosphere soil of tea trees in these production areas. The results showed that the weathered black rock soil in some areas had high total and available selenium content, and tea leaves grown on this soil had even higher selenium content. Selenium tolerance was screened for microorganisms in the weathered black rock soil of tea tree rhizospheres, and the selected selenium-tolerant strains were identified molecularly. This led to the discovery of the preserved Bacillus megaterium NT2 strain. 。

[0018] The Bacillus megaterium NT2 strain screened in this invention has a strong selenium reducing ability. According to Se... 4+ The screening results showed that Bacillus megaterium NT2 had a total selenium content of 9144.08±250.82 μg / g CDW, a biomass of 1.46±0.04 g CDW / L, a selenium yield of 13350.36±530.09 μg / L, and a selenium conversion rate of 13350.36 μg / L÷20000 μg / L=66.75%, demonstrating a strong selenium reduction and enrichment ability. At the same time, the conversion rates of this strain for heavy metals As, Cd, and Pb were 19.58%, 20.54%, and 19.08%, respectively, indicating a weak ability to enrich arsenic, cadmium, and lead.

[0019] The Bacillus megaterium NT2 strain screened in this invention exhibits strong selenium reduction and enrichment capabilities in high-temperature calcined activated rock powder, but its enrichment capabilities for heavy metals are relatively low. Selenium in high-selenium rocks is primarily found within the mineral lattice, resulting in low selenium availability. When high-selenium rock powder is calcined together with the calcination additive CaCO3, during the temperature rise, the carbonaceous matter and other easily oxidized components in the high-selenium rock react with oxygen and are oxidized. CaCO3 reacts with the mineral sample upon heating, producing a eutectic, which lowers the mineral decomposition temperature. Simultaneously, the generated CO2 is gradually released, leading to a porous structure in the mineral sample. This structure facilitates contact between oxygen and CaCO3 and the mineral, accelerating the reaction and causing the conversion of low-valence selenium to soluble high-valence selenium, releasing the rock selenium and increasing its effective selenium activation. The CaO produced by the thermal decomposition of CaCO3 acts as a selenium adsorbent, combining with the selenium oxide SeO2 produced during the calcination of high-selenium rocks to produce CaSeO4, effectively reducing selenium volatilization. The generated selenate (CaSeO4) is adsorbed on the surface of porous minerals. During phosphate extraction, competitive adsorption occurs, and the selenate is replaced by phosphate and released into the solution, thus improving the extraction efficiency of selenium speciation from rocks.

[0020] This invention involves adding high-temperature calcined activated rock powder to the LB liquid medium of Bacillus megaterium NT2. After testing, the selenium concentration in the culture medium was 18945.22 μg / L, the total selenium content of strain NT2 was 10058.49±295.97 μg / g CDW, the biomass was 1.55±0.08 g CDW / L, the selenium yield was 15590.65±530.09 μg / L, and the selenium conversion rate was 15590.65 μg / L ÷ 18945.22 μg / L = 82.29%, demonstrating a strong selenium enrichment and reduction ability. The total contents of heavy metals As, Cd, and Pb in strain NT2 were 0.47, 0.11, and 0.24 μg / g, respectively. The conversion rates of heavy metals As, Cd, and Pb by the bacterial cells were 19.58%, 20.54%, and 19.08%, respectively, which were lower than the conversion rates of selenium. This will effectively control the heavy metal content in the mixture of nano-selenium and bacterial cells.

[0021] The inventors simultaneously screened two strains of Bacillus megaterium, NG and NT. High-temperature calcined activated rock powder was added to the LB liquid medium of Bacillus megaterium NG. After testing, the Se concentration in the culture medium was 19054.41±784.43 μg / L, the total Se content of Bacillus megaterium NG was 9682.16±279.23 μg / g CDW, the biomass was 1.44±0.08 g CDW / L, the selenium yield was 13928.60±346.40 μg / L, and the Se conversion rate was =13928.60 μg / L ÷ 19054.41 μg / L = 73.10%.

[0022] High-temperature calcined activated rock powder was added to LB liquid medium of Bacillus megaterium NT. The Se concentration in the culture medium was 14164.23±694.76 μg / L, the total Se content of strain NT2 was 7353.37±262.87 μg / g CDW, the biomass was 1.66±0.10 g CDW / L, the Se yield was 12167.81±430.58 μg / L, and the Se conversion rate was =12167.81 μg / L ÷ 14164.23 μg / L = 85.91%. Finally, the nano-selenium organic fertilizer prepared by the present invention using a mixture of NT2 strain and nano-selenium can significantly increase the selenium content in tea. Experimental results show that, 1.5 to 4.5 months after root application of activated high-selenium rock powder organic fertilizer and nano-selenium organic fertilizer to tea gardens, the selenium content of tea increased from below the lower limit of selenium content in selenium-enriched tea to the standard for selenium-enriched tea. Furthermore, the selenium content of tea treated with nano-selenium organic fertilizer increased by 55.10% to 88.89% compared to tea treated with activated high-selenium rock powder organic fertilizer. Meanwhile, the lead and cadmium contents of tea treated with nano-selenium organic fertilizer did not increase significantly and did not exceed the standards. Therefore, nano-selenium reduced by Bacillus megaterium NT2 has higher selenium bioavailability, can effectively increase the selenium content of tea, and does not significantly change the heavy metal content. Attached Figure Description

[0023] Figure 1 This is a colony morphology diagram of Bacillus megaterium NT2 LB medium preserved in this invention; Figure 2 Colony morphology of Bacillus megaterium NT, NT2, and NG on selenium-containing culture medium; Figure 3 To plot the selenium standard curve using inductively coupled plasma atomic emission spectrometry. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0025] The required experimental instruments mainly include: a clean bench to provide a sterile operating environment; a constant temperature shaker to simulate the natural shaking during biological culture; a high-speed centrifuge to separate different components in a sample; an inductively coupled plasma atomic emission spectrometer to determine the concentration of specific substances in a solution; a PCR instrument to amplify specific DNA sequences; an electrophoresis apparatus to separate and analyze nucleic acid molecules; and a gel image analysis system to record and analyze electrophoresis results.

[0026] LB liquid medium: LB liquid medium is used for strain activation, expansion culture and shake flask fermentation. Accurately weigh 5 g / L yeast powder, 10 g / L NaCl and 10 g / L tryptone according to the required amount, add ddH2O to make up to the required mark, and sterilize by moist heat at 121℃ for 30 min.

[0027] 200 mg / mL Se 4+ Mother liquor: Accurately weigh 4.38 g of sodium selenite, add ddH2O to make up to 10 mL, filter through a membrane to remove impurities, and store at 4℃ for later use.

[0028] 20 mg / mL Se 4+ Stock solution: Take 1 mL of 200 mg / mL Se 4+ The stock solution was diluted to 10 mL with ddH2O and stored at 4°C for later use.

[0029] In this embodiment, the determination of total selenium content refers to the method for total selenium detection in the national standard GB / T 39356-2020 "Determination of total nickel, total cobalt, total selenium, total vanadium, total antimony and total thallium content in fertilizers by inductively coupled plasma atomic emission spectrometry". Example

[0030] In this embodiment, a strain of Bacillus megaterium NT2 was isolated from weathered soil of black high-selenium rock in Ankang, Shaanxi Province, and deposited at the China Center for Type Culture Collection on August 8, 2025. Latin name: Priestia megaterium NT2, strain preservation number CCTCC NO: M 20251806, address: Wuhan University, Wuhan, Hubei Province, postcode: 430072, telephone: 027-68754052.

[0031] The colonies of Bacillus megaterium NT2 preserved in this invention are round, such as... Figure 1 As shown, the mycelium appears pale yellowish-white on LB medium, with neat edges and a slightly glossy surface; on selenium-containing medium, it appears red (see...). Figure 2 The inventors first studied the selenium content of tea leaves in Ziyang County, Ankang, Shaanxi Province. They discovered that tea leaves with high selenium content were concentrated in certain areas, where the soil was mostly high-selenium black weathered rock soil. Therefore, they sampled and analyzed the black weathered rock soil, studying the microorganisms within it, and using a gradient concentration of Se... 4+LB agar plates were used for screening, and the selected selenium-resistant strains were identified molecularly. Simultaneously, bacterial DNA was amplified by PCR using primer pair 27F / 1492R, yielding a 16S rDNA gene sequence of approximately 1.5 kb. The amplification products were analyzed by 1% agarose gel electrophoresis, and clear electrophoretic images were obtained under a UV imaging system, observed, and preserved. The PCR products were then gel-cleaved and recovered using the AxyPrep DNA gel extraction kit and sent to the Xi'an Qingke Sequencing Center for sequencing. The sequencing results were compared with known sequences in the database to identify the bacterial species.

[0032] PCR amplification of strain NT2 DNA was performed using primer pair 27F / 1492R to obtain the 16S rDNA gene sequence. The PCR reaction system was prepared strictly according to the instructions of the high-fidelity DNA polymerase kit. 30 amplification cycles were set in the PCR instrument, and the annealing temperature was optimized to 55℃ to ensure amplification specificity. To verify the successful amplification of the target gene fragment, the PCR product was mixed with 6×DNA Loading Buffer and electrophoretically separated on a 1% (w / v) agarose gel (containing 0.5 μg / mL GelRed nucleic acid dye). After electrophoresis at a constant voltage of 120V for 30 minutes, the gel was transferred to a fully automated gel imaging system for UV detection. The results showed that the strain exhibited a single, bright, specific band in a region of approximately 1500 bp, highly consistent with the expected 16S rDNA amplification fragment length. No non-specific amplification bands were observed in the negative control group, indicating that this PCR amplification had good specificity and reproducibility. Sequencing analysis and comparison identified NT2 as belonging to Bacillus megaterium (Betatomium megaterium). Bacillus magaterium ).

[0033] NT2 16S rRNA sequence information: Example

[0034] This embodiment provides a screening method for Bacillus megaterium NT2, as follows: Different selenium-tolerant bacteria screened from weathered black rock soil in tea gardens will be inoculated with different concentrations of Se. 4+ After culturing in LB medium for a certain period of time, the bacterial cells were collected, and the selenium content, selenium conversion rate and biomass of the bacterial cells were measured. The differences between different strains were compared, and nano-selenium conversion strains with strong selenium accumulation ability and high biomass were screened.

[0035] Bacterial strain culture: The bacterial strain was inoculated into 3 mL of LB liquid medium and cultured at 37℃ and 200 rpm for 16 h to obtain primary seed culture; 0.5 mL of the primary seed culture was inoculated into 5 mL of LB liquid medium and cultured at 37℃ and 200 rpm for 16 h to obtain secondary seed culture; all 5 mL of the secondary seed culture was inoculated into a medium containing 20 μg / mL Se. 4+ The culture was carried out in 50 mL of LB liquid medium at 37°C and 200 rpm for 30 h to produce the selenium conversion fermentation broth.

[0036] (2) Strain collection: Pour 50 mL of fermentation broth into a centrifuge tube, centrifuge at 8000 rpm for 5 min, retain the bacterial cells, and discard the supernatant; add 3 mL of ddH2O to resuspend the bacterial cells, transfer the bacterial cells to a 10 mL centrifuge tube (weighed beforehand and recorded as empty tube weight), centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the red precipitate. Add 3 mL of ddH2O again to resuspend the bacterial cells, transfer the bacterial cells to a 10 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, collect the red precipitate, freeze-dry, and obtain a mixture of nano-selenium and bacterial cells powder, and record the total weight.

[0037] (3) Detection of total selenium content in bacteria: Refer to the method for total selenium detection in the national standard GB / T 39356-2020 "Determination of total nickel, total cobalt, total selenium, total vanadium, total antimony and total thallium content in fertilizers by inductively coupled plasma atomic emission spectrometry".

[0038] (4) Calculation of total selenium production and selenium conversion rate: Total selenium yield (μg / L) = Selenium content (μg / g CDW) × Biomass (g CDW / L) Selenium conversion rate = Total selenium yield (μg / L) / Initial Selenium content 4+ Concentration (μg / L) × 100% The results showed that the total Se content of strain NT2 was 9144.08±250.82 μg / g CDW, the biomass was 1.46±0.04 g CDW / L, the Se yield was 13350.36±530.09 μg / L, and the Se conversion rate was 66.75%. Compared with other similar strains, it showed a strong selenium enrichment and conversion ability. Example

[0039] In this embodiment, Bacillus megaterium strain NT2 was combined with high-selenium rocks from Ziyang County, Ankang City, Shaanxi Province to prepare a mixture of nano-selenium and bacterial cells.

[0040] The high-selenium rocks selected in this embodiment from Haoping Town, Ziyang County, Ankang City, Shaanxi Province, are all carbonaceous slates, characterized by a grayish-black appearance, a C content of 14.31±1.77%, a S content of 3.18±0.34%, a Se content of 78.50±10.71 mg / kg, and heavy metal contents of As, Cd, and Pb of 12.78±1.62 mg / kg, 2.66±1.53 mg / kg, and 6.56±0.61 mg / kg, respectively. See Table 1.

[0041]

[0042] Elements that can be absorbed by plant roots during the plant's growth period are called bioavailable elements. Bioavailable selenium and arsenic in rocks are extracted using a 0.1 mol / L KH₂PO₄-K₂HPO₄ solution: Weigh 1.0000 g of sample, add 10 mL of 0.10 mol / L KH₂PO₄-K₂HPO₄ solution, shake at 250 rpm for 240 min, centrifuge at 4000 rpm for 10 min, then add 10 mL of ultrapure water and repeat once. Combine the two supernatants for later use. Bioavailable lead and cadmium are extracted using a 0.005 mol / L DTPA - 0.1 mol / L TEA - 0.01 mol / L CaCl₂ solution: Weigh 10.00 g of sample, add 20.0 mL of DTPA-TEA-CaCl₂ extractant, shake at 180 rpm for 120 min, centrifuge at 4000 rpm for 10 min, and collect the supernatant for later use. The available selenium content of Se, As, Cd, and Pb in the Ziyang high-selenium rocks was 5.89%±2.06%, 0.22%±0.02%, 0.99%±0.20%, and 3.44%±0.45%, respectively. (See Table 2.)

[0043]

[0044] Methods for activating high-selenium rocks: In high-selenium rocks, selenium is mainly found within the mineral crystal lattice, resulting in low selenium availability. When high-selenium rock powder is roasted together with the roasting additive CaCO3, easily oxidizable components such as carbonaceous and sulfurous substances in the high-selenium rock react with oxygen and volatilize during the temperature rise. CaCO3 reacts with the mineral sample upon heating, producing a eutectic, which lowers the mineral decomposition temperature. Simultaneously, the generated CO2 is gradually released, leading to a porous structure in the mineral sample. This structure facilitates contact between oxygen and CaCO3 and the mineral, accelerating the reaction and causing the conversion of low-valence selenium to soluble high-valence selenium, releasing selenium from the rock and increasing the activation of available selenium. The thermal decomposition of CaCO3 produces CaO and CO2 (volatilization). CaO acts as a selenium adsorbent and can combine with selenium oxide (SeO2) produced during the roasting of high-selenium rocks to produce CaSeO4, thereby reducing selenium volatilization. The volatility of SeO2 produced in this process is 12.5±2.5%, and the carbon and sulfur (C+S) content is 18.19%±0.94%. The theoretical value of the total selenium content of the high-selenium rock powder after roasting should be 78.50mg / kg×(1-0.125)÷(1-0.1819)=83.96mg / kg.

[0045] After high-temperature roasting, the contents of Se, As, Cd, and Pb in the high-selenium rock were 79.62±12.98 mg / kg, 14.40±1.73 mg / kg, 2.91±1.48 mg / kg, and 7.57±0.66 mg / kg, respectively. The selenium availability increased from 5.89% before roasting to 83.72% after roasting, the as availability increased from 0.22% to 0.58%, the Cd availability increased from 0.99% to 1.28%, and the Pb availability decreased from 3.44% to 2.67% (see Table 2), indicating that the availability of heavy metals was not significantly activated after roasting.

[0046] This embodiment provides a specific method for preparing a mixture of nano-selenium and bacterial cells as follows.

[0047] (1) Activation of high-selenium rocks: The optimized roasting conditions for high-selenium rocks were determined by single-factor analysis and response surface methodology: high-selenium rock powder particle size: 40 mesh; roasting additive: CaCO3; roasting additive ratio (mass ratio) = high-selenium rock: CaCO3 = 1:0.4; roasting temperature: 400~600℃; roasting time: 3~5h. The proportion of selenium available in the high-selenium rocks increased from 5.89% before roasting to 83.72% after roasting.

[0048] (2) Preparation of high-selenium concentration liquid culture medium: Activated high-selenium rock powder was passed through a 400-mesh sieve and added to LB liquid culture medium (rock-liquid mass-to-volume ratio 1:4). The medium was shaken at 200 rpm for 12 h and allowed to stand for 8 h before the high-selenium rock powder settled naturally to obtain a high-selenium concentration culture medium. The culture medium had a selenium concentration of 18945.22 μg / L, an As concentration of 3.72 μg / L, a Cd concentration of 0.83 μg / L, and a Pb concentration of 1.95 μg / L.

[0049] (3) Culture of NT2 fermentation broth: Select a single clonal strain of NT2 and inoculate it into 3 mL of LB liquid medium. Culture at 37℃ and 200 rpm for 16 h to obtain primary seed liquid. Take 0.5 mL of primary seed liquid and inoculate it into 5 mL of LB liquid medium. Culture at 37℃ and 200 rpm for 16 h to obtain secondary seed liquid. Inoculate the secondary seed liquid at 10% in the prepared high selenium concentration liquid culture medium (2). Culture at 37℃ and 200 rpm for 30 h to obtain selenium conversion fermentation broth.

[0050] (4) Collection of nano-selenium and bacterial mixture: The fermentation broth was centrifuged at 8000 rpm for 5 min to collect the precipitate. The bacterial cells were then resuspended in ddH2O and centrifuged at 8000 rpm for 5 min to collect the red precipitate. The red precipitate was then washed with ddH2O and freeze-dried to obtain nano-selenium and bacterial mixture powder, which is the selenium source for nano-selenium organic fertilizer.

[0051] (5) The total selenium and heavy metal content of the mixture of nano-selenium and bacterial cells were tested using the national standard method.

[0052] A: Selenium content of the mixture of nano-selenium and bacterial cells: The selenium concentration in the culture medium was 18945.22 μg / L, the total selenium content of strain NT2 was 10058.49±295.97 μg / g CDW, the biomass was 1.55±0.08 g CDW / L, the selenium yield was 15590.65±530.09 μg / L, and the selenium conversion rate was 15590.65 μg / L ÷ 18945.22 μg / L = 82.29%, showing a strong selenium enrichment and reduction ability.

[0053] B: Heavy metal content of the mixture of nano-selenium and bacterial cells: The total contents of heavy metals As, Cd, and Pb in strain NT2 were 0.47, 0.11, and 0.24 μg / g, respectively. The conversion rates of heavy metals As, Cd, and Pb by the bacterial cells were 19.58%, 20.54%, and 19.08%, respectively. The conversion rates of heavy metals by the bacterial cells were lower than those of Se.

[0054] Using the same high-selenium rocks and the same method, and with Bacillus megaterium NG and NT isolated by the inventors as controls, the total selenium and heavy metal content were detected using the national standard method. The results are as follows.

[0055] Bacillus megaterium NG A: Selenium content of the mixture of nano-selenium and bacterial cells: the selenium concentration in the culture medium was 19054.41±784.43μg / L, the total selenium content of Bacillus megaterium NG was 9682.16±279.23μg / g CDW, the biomass was 1.44±0.08 CDW / L, the selenium yield was 13928.60±346.40μg / L, and the selenium conversion rate was 13928.60μg / L÷19054.41μg / L = 73.10%, showing a strong selenium enrichment and reduction ability.

[0056] B: Heavy metal content of the mixture of nano-selenium and bacterial cells: The total contents of heavy metals As, Cd, and Pb in strain NG were 1.79, 0.49, and 0.88 μg / g, respectively. The conversion rates of heavy metals As, Cd, and Pb by the bacterial cells were 69.29%, 85.01%, and 64.98%, respectively. The bacterial cells had a high enrichment capacity for heavy metals, with a higher enrichment capacity for Cd than for Se.

[0057] Bacillus megaterium NT A: Selenium content of the mixture of nano-selenium and bacterial cells: the selenium concentration in the culture medium was 14164.23±694.76μg / L, the total selenium content of strain NT2 was 7353.37±262.87μg / g CDW, the biomass was 1.66±0.10 g CDW / L, the selenium yield was 12167.81±430.58μg / L, and the selenium conversion rate was 12167.81μg / L÷14164.23μg / L = 85.91%, showing a strong selenium enrichment and reduction ability.

[0058] B: Heavy metal content of the mixture of nano-selenium and bacterial cells: The total contents of heavy metals As, Cd, and Pb in strain NT were 1.92, 0.46, and 0.91 μg / g, respectively. The conversion rates of heavy metals As, Cd, and Pb by the bacterial cells were 85.68%, 92.00%, and 77.47%, respectively. The bacterial cells had a high enrichment capacity for heavy metals, with a higher enrichment capacity for As and Cd than for Se. Example

[0059] This embodiment describes the preparation of a nano-selenium organic fertilizer based on the preserved Bacillus megaterium NT2 strain and the nano-selenium and bacterial mixture prepared from activated high-selenium rock powder. The specific preparation method is as follows.

[0060] ① Crush dried auxiliary materials such as soybean cake, rapeseed cake and straw, and mix them evenly with animal manure and auxiliary materials to adjust the C / N ratio in a certain proportion, controlling the carbon-nitrogen ratio to be 25:1 to 30:1, to obtain an organic matter mixture.

[0061] ② Spray the fermentation broth of Bacillus megaterium NT2 evenly onto the organic matter mixture at a mass ratio of 1-5%, and adjust the moisture content of the mixture to 50%-60% with water. ④ Pile the materials into a pile 1-1.5 meters high and 1.5-2 meters wide, and cover it with straw mats to ensure the temperature is between 28-37℃ and the humidity is between 60-80%.

[0062] ⑤ When the temperature of the fermentation pile rises above 65°C and begins to drop and stabilize below 50°C, thoroughly turn the pile over. At the same time, add 1-1.5% of a mixture of nano-selenium and microbial cells evenly to the fermentation pile as a selenium source and continue fermentation.

[0063] ⑥ Composting Management and Testing: When the pile temperature drops to ambient temperature and remains stable, and the material is dark brown, loose, odorless, and has an earthy aroma, composting is complete. After the organic fertilizer composition and selenium content meet the standards, nano-selenium organic fertilizer is obtained.

[0064]

[0065] The aforementioned nano-selenium organic fertilizer was applied to tea gardens, with the control group receiving no treatment. The treatment groups were treated with activated high-selenium rock powder organic fertilizer and nano-selenium organic fertilizer, respectively. Fertilization studies were conducted at multiple tea garden sites in Ankang. Selenium content was measured in tea leaves (one bud and two leaves) harvested in January / May, March, and April / May. (See Table 4). The results showed that before selenium application, the selenium content of tea leaves in both experimental areas was below 0.10 mg / kg, failing to meet the standard for selenium-enriched tea. 1.5 months after selenium application, the selenium content in the T1 (activated high-selenium rock powder organic fertilizer) treatment in both experimental areas was 0.18~0.25 mg / kg, and the selenium content in the T2 (nano-selenium organic fertilizer) treatment was 0.34~0.42 mg / kg; 3 months after selenium application, the selenium content in the T1 (activated high-selenium rock powder organic fertilizer) treatment in both experimental areas was 0.44~0.49 mg / kg, and the selenium content in the T2 (nano-selenium organic fertilizer) treatment was 0.69~0.76 mg / kg; 4.5 months after selenium application, the selenium content in the T1 (activated high-selenium rock powder organic fertilizer) treatment in both experimental areas was 0.71~0.74 mg / kg, and the selenium content in the T2 (nano-selenium organic fertilizer) treatment was 1.24~1.29 mg / kg. After selenium application, the selenium content of tea leaves increased from below the lower limit of selenium content in selenium-enriched tea to the standard level. Furthermore, the selenium content of tea leaves treated with T2 (nano-selenium organic fertilizer) increased by 55.10% to 88.89% compared to those treated with T1 (activated high-selenium rock powder organic fertilizer). This demonstrates that nano-selenium reduced by Bacillus megaterium NT2 has higher selenium bioavailability and can effectively increase the selenium content of tea leaves.

[0066] The Pb, Cd, and As contents in tea were tested according to national standards GB 5009.12, GB 5009.15, and GB 5009.11. The Pb, Cd, and As contents in the control group tea were 0.28~0.44 mg / kg, 0.019~0.044 mg / kg, and 0.055~0.092 mg / kg, respectively. The Pb, Cd, and As contents in the T2 (nano-selenium organic fertilizer) treated tea were 0.22~0.46 mg / kg, 0.017~0.038 mg / kg, and 0.046~0.11 mg / kg, respectively. It can be seen that the nano-selenium organic fertilizer prepared using this Bacillus megaterium did not significantly increase the Pb, Cd, and As contents in tea. According to GB 2762-2022 "National Food Safety Standard - Limits of Contaminants in Food", the lead content in tea is ≤5.0 mg / kg. According to NY 659-2003 "Limits of Chromium, Cadmium, Mercury, Arsenic and Fluorides in Tea", the cadmium content in tea is ≤1 mg / kg and the arsenic content is ≤2 mg / kg. After applying nano-selenium organic fertilizer, the Pb, Cd and As content in tea did not exceed the standards.

[0067]

Claims

1. A type of Bacillus megaterium, characterized in that: The Latin name of the aforementioned Bacillus megaterium: Priestia megaterium NT2 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20251806.

2. The use of the Bacillus megaterium according to claim 1, characterized in that: The Bacillus megaterium NT2 reduces inorganic selenium to nano-selenium under the premise of low arsenic, cadmium, and lead conversion rates.

3. The use of Bacillus megaterium according to claim 2, characterized in that: The Bacillus megaterium NT2 bacterial solution was mixed with high-selenium rock powder activated by high-temperature calcination and fermentation to obtain a mixture of nano-selenium and bacterial cells, and this mixture was used as a selenium source to prepare nano-selenium organic fertilizer.

4. A method for preparing the mixture of nano-selenium and bacterial cells as described in claim 3, characterized in that, Includes the following steps: ① Activation of high-selenium rocks: Mix high-selenium rock powder with CaCO3 and calcine at 400~600℃ for 3~5 hours to obtain activated high-selenium rock powder; ② Preparation of high-selenium rock culture medium: Add activated high-selenium rock powder to LB liquid culture medium, shake, and let stand until the high-selenium rock powder settles naturally to obtain high-selenium rock culture medium; ③ Culture of the strain: The cultured Bacillus megaterium NT2 fermentation broth was inoculated into the prepared high-selenium rock culture medium and aerobic fermentation was carried out to obtain selenium conversion fermentation broth; the preservation number of Bacillus megaterium NT2 is: CCTCC NO: M20251806; ④ Collection of nano-selenium and bacterial cell mixture: Centrifuge the fermentation broth to collect the precipitate, then add ddH2O to resuspend the bacterial cells, centrifuge the bacterial cells a second time to collect the red precipitate, wash the red precipitate with ddH2O and freeze-dry to obtain nano-selenium and bacterial cell mixture powder.

5. The method for preparing the mixture of nano-selenium and bacterial cells according to claim 4, characterized in that: The mass ratio of the high-selenium rock powder to CaCO3 is 1:0.

4.

6. The method for preparing the mixture of nano-selenium and bacterial cells according to claim 4, characterized in that: The aerobic fermentation is specifically carried out at 37±2℃ and 100~300 rpm for 24~48 hours.

7. The method for preparing the mixture of nano-selenium and bacterial cells according to claim 4, characterized in that: The activated high-selenium rock powder and LB liquid culture medium were in a mass-to-volume ratio of 1:

4.

8. A method for preparing nano-selenium organic fertilizer using the nano-selenium and microbial mixture as described in claim 4, characterized in that, Includes the following steps: ① Crush soybean cake, rapeseed cake and dried straw auxiliary materials, mix them evenly with animal manure, and control the carbon-nitrogen ratio to be 25:1~30:1 to obtain an organic matter mixture; ② Spray the fermentation broth of Bacillus megaterium NT2 evenly onto the organic matter mixture at a mass ratio of 1~5%, and adjust the water content of the organic matter mixture to 50%~60% with water. The preservation number of Bacillus megaterium NT2 is: CCTCC NO: M20251806. ③Pile the material in a warm, moist, and aerobic environment with a temperature of 28~37℃ and a humidity of 60%~80%; ④ When turning the fermentation pile, add 1~1.5% of a mixture of nano-selenium and microbial cells evenly to the fermentation pile as a selenium source and continue fermentation; ⑤ After fermentation and decomposition, the organic fertilizer components and selenium content are tested and found to meet the standards. Once these conditions are met, fermentation is stopped to obtain nano-selenium organic fertilizer.

9. The use of the Bacillus megaterium according to claim 1, characterized in that: The Bacillus megaterium NT2 was applied to tea trees to increase the selenium content in tea leaves.

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