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

Abstract

The application provides a bacillus megaterium and application of the bacillus megaterium in preparation of nano selenium organic fertilizer for increasing selenium content of tea, and belongs to the technical field of microorganisms, wherein the bacillus megaterium NT2 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:M 20251806; the bacillus megaterium NT2 has strong tolerance and reduction capacity to selenium elements, can survive in high-concentration inorganic selenium, and reduce the inorganic selenium into nano selenium, and has weak capacity to enrich arsenic, cadmium and lead; can be mixed with high-temperature calcination activated high-selenium rock powder to carry out mixed fermentation, to prepare a selenium source of nano selenium organic fertilizer, to increase the selenium content of tea while ensuring that the heavy metal content does not exceed the national standard. The bacillus megaterium provided by the application can reduce the toxicity of inorganic selenium in high-selenium rock and convert it into nano selenium, and can stably, safely and standardly produce selenium-rich tea, so that the heavy metal content of tea does not exceed the standard, and the application has a broad 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 area is less than 26%, and the selenium content of most tea is less than 0.25 mg / kg, the main reason is the point distribution and uneven distribution of soil selenium content, which leads to the instability of tea selenium content. Therefore, using soil microbial inoculant to promote plant absorption of soil selenium or using microorganisms to transform 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 not having high reduction and enrichment ability for arsenic, cadmium and lead, and nano selenium organic fertilizer is prepared using the nano selenium to root tea trees to ensure that the selenium content of tea is stable to reach the standard of selenium-rich tea, and the heavy metal content of tea is not increased.

[0007] The present application first selects a strain of Bacillus megaterium NT2, which is preserved in China Typical Culture Collection Center on August 8, 2025, and the Latin name is: Priestia megaterium NT2, the strain preservation 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 preserved Bacillus megaterium NT2 of the present application is round, as shown in Figure 1 , the color on the LB medium is light yellowish white, the edge of the bacterial lawn is neat, and the surface is slightly glossy; 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]

[0011] The bacillus megaterium NT2 preserved based on the application has strong selenium reduction capacity, and can reduce inorganic selenium into nano selenium. The nano selenium has higher bioavailability and lower toxicity than inorganic selenium. The bacillus megaterium NT2 can be used to reduce inorganic selenium into nano selenium and bacterial body mixture, so as to reduce the toxicity of inorganic selenium and improve the bioavailability of selenium. The nano selenium organic fertilizer prepared by using the nano selenium and bacterial body mixture as the selenium source is applied to the root of tea tree, so as to improve the selenium content in tea leaves, and the heavy metal content in tea leaves has no significant change compared with the control.

[0012] Therefore, the bacillus megaterium NT2 can be used to prepare the nano selenium organic fertilizer for improving the selenium content in tea leaves.

[0013] The application further provides a preparation method of the nano selenium and bacterial body mixture, comprising the following steps:

[0014] ① high selenium rock activation: the high selenium rock powder is mixed with CaCO3, and is calcined at 400-600 DEG C for 3-5 h to obtain the activated high selenium rock powder;

[0015] ② preparation of high selenium rock culture solution: the activated high selenium rock powder is passed through 400 meshes and is added into LB liquid medium, and after oscillation treatment, the high selenium rock culture solution is obtained by static treatment until the high selenium rock powder is naturally settled;

[0016] ③ culture of the strain: the fermented liquid of the cultured bacillus megaterium NT2 is inoculated into the prepared high selenium rock culture solution, and aerobic fermentation is carried out to obtain selenium conversion fermented liquid; the preservation number of the bacillus megaterium NT2 is CCTCC NO: M20251806;

[0017] ④ collection of the nano selenium and bacterial body mixture: the precipitate is collected by centrifugation of the fermented liquid, the bacterial body is resuspended by adding ddH2O, the bacterial body is centrifuged again, the red precipitate is collected, the red precipitate is washed by blowing ddH2O, and then freeze-drying is carried out to obtain the nano selenium and bacterial body mixture powder.

[0018] The mixing mass ratio of the high selenium rock powder and CaCO3 is 1:0.4.

[0019] The aerobic fermentation is specifically carried out at 37±2 DEG C and 100-300 rpm for 24-48 h.

[0020] The mass-volume ratio of the activated high selenium rock powder and LB liquid medium is 1:4.

[0021] Meanwhile, the application further provides a preparation method of the nano selenium organic fertilizer by using the nano selenium and bacterial body mixture as the selenium source, comprising the following steps:

[0022] ① bean cake, rapeseed cake and straw dry auxiliary are crushed, mixed with animal manure uniformly, and the carbon-nitrogen ratio is controlled to be 25:1~30:1, so that the organic matter mixture is obtained;

[0023] ② 1~5% of the fermentation liquor of bacillus megaterium NT2 is sprayed on the organic matter mixture according to the mass ratio, and the water content of the organic matter mixture is adjusted to 50%~60% by using water, wherein the preservation number of the bacillus megaterium NT2 is CCTCC NO: M20251806;

[0024] ③ The material is stacked and oxygen fermentation is carried out under the condition that the temperature is 28~37 DEG C and the humidity is 60~80%;

[0025] ④ 1~1.5% of the nano selenium and bacterial body mixture are added uniformly in the fermentation pile as a selenium source during fermentation and turning over, and the fermentation is continued;

[0026] ⑤ After fermentation and maturation, the organic fertilizer composition and selenium content are detected, the fermentation is stopped when the standards are reached, and the nano selenium organic fertilizer is obtained. Beneficial effects

[0027] The inventor investigates and studies the selenium content of tea leaves in Ziyang County, Ankang, Shaanxi, delimits the natural selenium-rich tea leaf (selenium content is greater than or equal to 0.25 mg / kg) production area, detects the total selenium and available selenium of the rhizosphere soil of the tea tree in the natural selenium-rich tea production area, finds that the black rock weathered soil in some areas has relatively high total selenium content and available selenium content, and the tea tree tea leaves grown in the black rock weathered soil have higher selenium content. Through selenium tolerance screening research on the microorganisms in the black rock weathered soil of the tea tree rhizosphere, and molecular biology identification of the screened selenium-resistant strains, the bacillus megaterium NT2 preserved in the application is obtained. 。

[0028] The bacillus megaterium NT2 screened in the application has strong selenium reduction capacity. According to Se 4+ According to the screening experiment results, the total selenium content of the bacillus megaterium NT2 is 9144.08±250.82 μg / g CDW, the biomass is 1.46±0.04 g CDW / L, the selenium yield is 13350.36±530.09 μg / L, and the selenium conversion rate is 13350.36 μg / L÷20000 μg / L=66.75%, which shows that the bacillus megaterium NT2 has strong selenium reduction and enrichment capacity. At the same time, the conversion rates of the bacillus megaterium NT2 to heavy metals As, Cd and Pb are 19.58%, 20.54% and 19.08% respectively, so the bacillus megaterium NT2 has weak enrichment capacity for arsenic, cadmium and lead.

[0029] The bacillus megaterium NT2 screened by the application also has strong selenium reduction and enrichment ability in the selenium in the high-temperature calcined and activated rock powder, and has low heavy metal enrichment ability. The selenium in the high-selenium rock mainly exists in the mineral lattice, and the selenium availability is low. When the high-selenium rock powder is roasted with the roasting additive CaCO3, the carbon and other easily oxidized components in the high-selenium rock will be oxidized first in the process of temperature rising. CaCO3 reacts with the mineral sample to produce a certain eutectic, which reduces the mineral decomposition temperature, and the generated CO2 is gradually released, so that the mineral sample forms a porous structure. This structure makes oxygen and CaCO3 more easily contact with the mineral to accelerate the reaction, so that the low-valence selenium is converted to soluble high-valence selenium, the rock selenium is released, and the effective selenium activation of the rock is increased. CaO produced by the thermal decomposition of CaCO3 is a selenium adsorbent, which combines with the selenium oxide SeO2 produced by the roasting of high-selenium rock to produce CaSeO4, which can effectively reduce the volatilization of selenium. The generated selenate (CaSeO4) is adsorbed on the surface of the porous mineral, and when phosphate is extracted, competitive adsorption occurs, and is replaced by phosphate to release into solution, thereby improving the extraction efficiency of the selenium form of the rock.

[0030] The application adds the high-temperature calcined and activated rock powder to the LB liquid medium of the bacillus megaterium NT2, and through detection, the selenium concentration of the culture solution is 18945.22 μg / L, the total selenium content of the strain NT2 is 10058.49±295.97 μg / g CDW, the biomass is 1.55±0.08 g CDW / L, the selenium yield is 15590.65±530.09 μg / L, and the selenium conversion rate is =15590.65 μg / L÷18945.22 μg / L = 82.29%, which shows strong selenium enrichment and reduction ability. The total content of heavy metals As, Cd and Pb in the strain NT2 is 0.47, 0.11 and 0.24 μg / g respectively. The conversion rate of the strain to heavy metals As, Cd and Pb is 19.58%, 20.54% and 19.08% respectively. The conversion rate of the strain to heavy metals is lower than that of selenium. This will effectively control the content of heavy metals in the mixture of nano-selenium and bacterial cells.

[0031] The inventors also screened two strains of bacillus megaterium NG and NT, and added the high-temperature calcined and activated rock powder to the LB liquid medium of the bacillus megaterium NG, and through detection, the selenium concentration of the culture solution is 19054.41±784.43 μg / L, the total selenium content of the bacillus megaterium NG is 9682.16±279.23 μg / g CDW, the biomass is 1.44±0.08 g CDW / L, the selenium yield is 13928.60±346.40 μg / L, and the selenium conversion rate is =13928.60 μg / L÷19054.41 μg / L = 73.10%.

[0032] The high-temperature calcined activated rock powder is added to the LB liquid culture medium of Bacillus megaterium NT, and through detection, the Se concentration of the culture solution is 14164.23±694.76 μg / L, the total Se content of the strain NT2 is 7353.37±262.87 μg / g CDW, the biomass is 1.66±0.10 g CDW / L, the Se yield is 12167.81±430.58 μg / L, and the Se conversion rate is =12167.81 μg / L÷14164.23 μg / L = 85.91%,

[0033] Finally, the nano-selenium organic fertilizer prepared by the NT2 strain and the nano-selenium mixture can significantly improve the selenium content in tea leaves. According to the experimental results, after the activated high-selenium rock powder organic fertilizer and the nano-selenium organic fertilizer are applied to the tea garden for 1.5 months to 4.5 months, the selenium content in tea leaves is increased from below the lower limit of the selenium content of selenium-rich tea to the standard of the selenium content of selenium-rich tea, and the selenium content in tea leaves treated by the nano-selenium organic fertilizer is increased by 55.10% to 88.89% compared with the selenium content in tea leaves treated by the activated high-selenium rock powder organic fertilizer. The lead and cadmium contents in tea leaves treated by the nano-selenium organic fertilizer do not increase significantly and do not exceed the standard. Therefore, the nano-selenium reduced by Bacillus megaterium NT2 has higher selenium bioavailability, can effectively improve the selenium content in tea leaves, and the heavy metal content does not change significantly. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The figure is a colony morphology diagram of Bacillus megaterium NT2 LB culture medium preserved by the application;

[0035] Figure 2 The figure is a colony morphology diagram of selenium-containing culture medium of Bacillus megaterium NT, NT2 and NG;

[0036] Figure 3 The figure is a standard curve of selenium prepared by inductively coupled plasma emission spectrometry. DETAILED DESCRIPTION

[0037] The technical solutions of the application will be further described in detail through specific examples and drawings.

[0038] The required experimental instruments mainly include: a super-clean workbench for providing a sterile operation environment; a constant-temperature shaking bed for simulating natural shaking in the biological culture process; a high-speed centrifuge for separating different components in the sample; an inductively coupled plasma emission spectrometer for determining the concentration of a specific substance in a solution; a PCR instrument for amplifying a specific DNA sequence; an electrophoresis instrument for separating and analyzing nucleic acid molecules; and a gel image analysis system for recording and analyzing electrophoresis results.

[0039] LB liquid medium: LB liquid medium was selected for strain activation, expansion culture and shake flask fermentation culture. 5 g / L yeast powder, 10 g / L NaCl and 10 g / L tryptone were accurately weighed according to the amount, ddH2O was added to the required scale, and 121℃ wet heat sterilization was performed for 30 min.

[0040] 200 mg / mL Se 4+ Stock solution: accurately weigh 4.38 g of sodium selenite, add ddH2O to 10 mL, filter membrane to remove impurities, and store at 4℃ for standby.

[0041] 20 mg / mL Se 4+ Stock solution: take 1 mL of 200 mg / mL Se 4+ Stock solution, add ddH2O to 10 mL, store at 4℃ for standby.

[0042] In the examples, the determination standard of total selenium content refers to the method for detecting total selenium 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 Inductively coupled plasma atomic emission spectrometry". Examples

[0043] In this example, a bacillus megaterium NT2 was isolated from the weathered soil of black high-selenium rock in Ankang, Shaanxi, and was preserved in the China Center for Type Culture Collection on August 8, 2025, with the Latin name: Priestia megaterium NT2, the strain preservation number is CCTCC NO: M 20251806, the address is Wuhan University, Wuhan, Hubei Province, China, the postcode is 430072, and the telephone number is 027-68754052.

[0044] The colony of the bacillus megaterium NT2 preserved in the application is circular, as shown in Figure 1 , the color on the LB medium is light yellow white, the edge of the bacterial lawn is neat, and the surface is slightly glossy; the color on the selenium-containing medium is red (see Figure 2 ). The inventors first studied the selenium content of tea in Ziyang County, Ankang, Shaanxi, and found that tea with high selenium content was concentrated in some areas, and the soil of which was mostly high-selenium black rock weathered soil. Therefore, the black rock weathered soil was sampled and analyzed, and the microorganisms therein were studied. Gradient concentration Se 4+LB solid plate screening, and the selected selenium-resistant strains were identified by molecular biology. At the same time, the primer pair 27F / 1492R was used to amplify the bacterial DNA by PCR, and the 16S rDNA gene sequence of about 1.5 kb was obtained. The amplification products were detected and analyzed by 1% agarose gel electrophoresis, and the clear electrophoretogram was obtained by imaging under the ultraviolet imaging system, and observed and saved. The PCR products were recovered by AxyPrep DNA gel recovery kit, and sent to Xi'an Jingke Sequencing Center for sequencing. The sequencing results were compared with the known sequences in the database to identify the bacterial species.

[0045] The primer pair 27F / 1492R was used to amplify the DNA of strain NT2 by PCR to obtain the 16S rDNA gene sequence. The PCR reaction system was strictly prepared according to the instructions of the high-fidelity DNA polymerase kit. Thirty amplification cycles were set in the PCR instrument, and the annealing temperature was optimized to 55°C to ensure the specificity of amplification. To verify the successful amplification of the target gene fragment, the PCR products were mixed with 6×DNA loading buffer, and then electrophoretically separated in 1% (w / v) agarose gel containing 0.5 μg / mL GelRed nucleic acid dye. After electrophoresis at a constant voltage of 120 V for 30 minutes, the gel was transferred to a full-automatic gel imaging system for ultraviolet light detection. The results showed that the strain presented a single, bright and specific band in the about 1500 bp region, which was highly consistent with the expected length of 16S rDNA amplification fragment. The negative control group did not appear non-specific amplification band, indicating that the PCR amplification had good specificity and repeatability. Sequencing analysis and comparison showed that NT2 belonged to Bacillus megaterium Bacillus magaterium ).

[0046] NT2 16S rRNA sequence information:

[0047] Examples

[0048] The present embodiment provides a screening method of Bacillus megaterium NT2, which is specifically as follows:

[0049] Different selenium-tolerant bacteria screened from black rock weathered soil in a tea garden were inoculated into LB medium containing different concentrations of Se 4+ , and after a certain period of culture, bacterial cells were collected, selenium content, selenium conversion rate and biomass were determined, and differences between different strains were compared to screen selenium-polymerizing strains with high biomass and high selenium conversion rate.

[0050] Strain culture: inoculate the bacterial strain into 3 mL of LB liquid medium, cultivate at 37°C and 200 rpm for 16 h as a primary seed; take 0.5 mL of the primary seed liquid, inoculate into 5 mL of LB liquid medium, cultivate at 37°C and 200 rpm for 16 h as a secondary seed; inoculate the 5 mL of the secondary seed liquid into 50 mL of LB liquid medium containing 20 μg / mL Se 4+ , cultivate at 37°C and 200 rpm for 30 h as a selenium conversion fermentation liquid.

[0051] (2) Strain collection: pour the 50 mL of fermentation liquid into a centrifuge tube, centrifuge at 8000 rpm for 5 min, leave the bacterial cells, and remove the supernatant; add 3 mL of ddH2O to resuspend the bacterial cells, transfer the bacterial cells to a 10 mL centrifuge tube (weighed in advance as the empty tube weight), centrifuge at 8000 rpm for 5 min, remove the supernatant, and collect the red precipitate. Resuspend the bacterial cells with 3 mL of ddH2O again, transfer the bacterial cells to a 10 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, remove the supernatant, and collect the red precipitate, freeze-dry, and obtain a mixture powder of nanoselenium and bacterial cells, and record the total weight.

[0052] (3) Total selenium content detection: 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 - Inductively Coupled Plasma Atomic Emission Spectrometry”.

[0053] (4) Total selenium yield and selenium conversion rate calculation:

[0054] Total selenium yield (μg / L) = selenium content (μg / g CDW) x biomass (g CDW / L)

[0055] Selenium conversion rate = total selenium yield (μg / L) / initial Se 4+ concentration (μg / L) x 100%

[0056] 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%, which showed a stronger selenium enrichment and conversion capacity compared with other similar strains. Example

[0057] In this embodiment, the Bacillus megaterium NT2 strain and high-selenium rocks in Ziyang County, Ankang City, Shaanxi Province are combined to prepare a mixture of nano selenium and bacterial cells.

[0058] In this embodiment, the high-selenium rocks in Haoping Town, Ziyang County, Ankang City, Shaanxi Province are all carbonaceous slate, with the appearance characteristics of gray-black color, C content of 14.31 ± 1.77%, S content of 3.18 ± 0.34%, Se content of 78.50 ± 10.71 mg / kg, and heavy metal As, Cd, and Pb contents of 12.78 ± 1.62 mg / kg, 2.66 ± 1.53 mg / kg, and 6.56 ± 0.61 mg / kg, respectively. See Table 1.

[0059]

[0060] The elements that can be absorbed by plant roots during the plant growth period are called bioavailable elements. The available state of selenium and arsenic in rocks is extracted by 0.1 mol / L KH2PO4-K2HPO4 solution: 1.0000 g of sample is added to 10 mL of 0.10 mol / L KH2PO4-K2HPO4 solution, oscillated at 250 rpm for 240 min, centrifuged at 4000 rpm for 10 min, and then 10 mL of ultrapure water is added and repeated once, and the two supernatants are combined for standby; the available state of lead and cadmium is extracted by 0.005 mol / L DTPA-0.1 mol / L TEA-0.01 mol / L CaCl2 solution: 10.00 g of sample is added to 20.0 mL of DTPA-TEA-CaCl2 extractant, oscillated at 180 rpm for 120 min, centrifuged at 4000 rpm for 10 min, and the supernatant is taken for standby. The available state of Se, As, Cd, and Pb in Ziyang high-selenium rocks accounts for 5.89% ± 2.06%, 0.22% ± 0.02%, 0.99% ± 0.20%, and 3.44% ± 0.45%, respectively. See Table 2.

[0061]

[0062] High-selenium rock activation method:

[0063] The selenium in high-selenium rock mainly exists in the mineral lattice, and the effectiveness of selenium is low. When the high-selenium rock powder is roasted together with the roasting additive CaCO3, the carbonaceous and sulfuraceous components in the high-selenium rock will first react with oxygen and volatilize during the temperature rising process. CaCO3 reacts with the ore sample to produce a certain eutectic, which reduces the mineral decomposition temperature, and the generated CO2 is gradually released, so that the ore sample forms a porous structure, which makes oxygen and CaCO3 more easily contact with the mineral to accelerate the reaction, so that the low-valence selenium is converted to soluble high-valence selenium, the selenium in the rock is released, and the effective selenium in the rock is activated and increased. CaCO3 is decomposed by heat to produce CaO and CO2 (volatilization), and the CaO is a selenium adsorbent that can produce CaSeO4 with the selenium oxide SeO2 produced by roasting of high-selenium rock, thereby reducing the volatilization of selenium. The volatilization rate of Se to produce SeO2 in this process is 12.5±2.5%, the carbon and sulfur (C+S) content is 18.19%±0.94%, and 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.

[0064] After high-temperature roasting, the contents of Se, As, Cd and Pb in the high-selenium rock are 79.62±12.98mg / kg, 14.40±1.73mg / kg, 2.91±1.48mg / kg and 7.57±0.66mg / kg respectively. The effectiveness of Se is increased from 5.89% before roasting to 83.72% after roasting, the effectiveness of As is increased from 0.22% before roasting to 0.58%, the effectiveness of Cd is increased from 0.99% before roasting to 1.28%, and the effectiveness of Pb is decreased from 3.44% before roasting to 2.67% (see Table 2), which indicates that the effective content of heavy metals is not largely activated after roasting.

[0065] The preparation method of the nano-selenium and bacteria mixture provided in this embodiment is as follows.

[0066] (1) High-selenium rock activation: through single factor analysis and response surface analysis, the optimized high-selenium rock roasting conditions are as follows: 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℃, and roasting time: 3-5h. The effective state ratio of selenium in high-selenium rock is increased from 5.89% before roasting to 83.72% after roasting.

[0067] (2) Preparation of high selenium concentration liquid medium: After the activated high selenium rock powder was passed through a 400 mesh sieve, it was added to LB liquid medium (rock powder: liquid volume ratio 1 : 4), shaken at 200 rpm for 12 h, and allowed to stand for 8 h. The high selenium rock powder naturally settled, and a high selenium concentration culture solution was obtained. The selenium concentration of the culture solution was 18945.22 μg / L, the As concentration was 3.72 μg / L, the Cd concentration was 0.83 μg / L, and the Pb concentration was 1.95 μg / L.

[0068] (3) Cultivation of NT2 fermentation broth: A single colony of strain NT2 was inoculated into 3 mL of LB liquid medium, and cultured at 37°C and 200 rpm for 16 h to obtain a primary seed solution. 0.5 mL of the primary seed solution was inoculated into 5 mL of LB liquid medium, and cultured at 37°C and 200 rpm for 16 h to obtain a secondary seed solution. The secondary seed solution was inoculated into the prepared high selenium concentration liquid medium (2) at a 10% inoculation amount, and cultured at 37°C and 200 rpm for 30 h to obtain a selenium conversion fermentation broth.

[0069] (4) Collection of nanometer selenium and bacterial mixture: The fermentation broth was centrifuged at 8000 rpm for 5 min to collect the precipitate, and the bacterial cells were resuspended in ddH2O. The bacterial cells were centrifuged at 8000 rpm for 5 min to collect the red precipitate. The red precipitate was washed with ddH2O and freeze-dried to obtain a nanometer selenium and bacterial mixture powder, which was the selenium source of the nanometer selenium organic fertilizer.

[0070] (5) The total selenium and heavy metal content of the nanometer selenium and bacterial mixture were detected by the national standard method.

[0071] A: The selenium content of the nanometer selenium and bacterial mixture: The selenium concentration of the culture solution 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 82.29%, indicating a strong selenium enrichment and reduction capacity.

[0072] B: The heavy metal content of the nanometer selenium and bacterial mixture: The total heavy metal content of strain NT2 was 0.47, 0.11, and 0.24 μg / g for As, Cd, and Pb, respectively. The conversion rate of the bacterial cells for heavy metals As, Cd, and Pb was 19.58%, 20.54%, and 19.08%, respectively, which was lower than that of Se.

[0073] The same high-selenium rock and the same method were used to detect the total selenium and heavy metal content in the mixture of nano-selenium and bacterial cells using Bacillus megaterium NG and NT isolated by the inventor as controls, and the results are as follows.

[0074] Bacillus megaterium NG

[0075] A: Selenium content of the mixture of nano-selenium and bacterial cells: the selenium concentration of the culture solution was 19054.41 ± 784.43 μg / L, the total selenium content of Bacillus megaterium NG was 9682.16 ± 279.23 μg / g CDW, the biomass was 1.44 ± 0.08 CDW / L, the selenium yield was 13928.60 ± 346.40 μg / L, and the selenium conversion rate was 13928.60 μg / L ÷ 19054.41 μg / L = 73.10%, which showed strong selenium enrichment and reduction capacity.

[0076] B: Heavy metal content of the mixture of nano-selenium and bacterial cells: the total content of heavy metals As, Cd, and Pb of strain NG was 1.79, 0.49, and 0.88 μg / g, respectively. The conversion rate of the bacterial cells to heavy metals As, Cd, and Pb was 69.29%, 85.01%, and 64.98%, respectively, and the bacterial cells had high enrichment capacity for heavy metals, with higher enrichment capacity for Cd than for Se.

[0077] Bacillus megaterium NT

[0078] A: Selenium content of the mixture of nano-selenium and bacterial cells: the selenium concentration of the culture solution was 14164.23 ± 694.76 μg / L, the total selenium content of Bacillus megaterium NT2 was 7353.37 ± 262.87 μg / g CDW, the biomass was 1.66 ± 0.10 g CDW / L, the selenium yield was 12167.81 ± 430.58 μg / L, and the selenium conversion rate was 12167.81 μg / L ÷ 14164.23 μg / L = 85.91%, which showed strong selenium enrichment and reduction capacity.

[0079] B: Heavy metal content of the mixture of nano-selenium and bacterial cells: the total content of heavy metals As, Cd, and Pb of strain NT was 1.92, 0.46, and 0.91 μg / g, respectively. The conversion rate of the bacterial cells to heavy metals As, Cd, and Pb was 85.68%, 92.00%, and 77.47%, respectively, and the bacterial cells had high enrichment capacity for heavy metals, with higher enrichment capacity for As and Cd than for Se. Example

[0080] Based on the preserved Bacillus megaterium NT2 strain and the mixture of nano-selenium and bacterial cells prepared from activated high-selenium rock powder described above, a nano-selenium organic fertilizer was prepared, and the specific preparation method is as follows.

[0081] ①The dry auxiliary materials such as bean cake, rapeseed cake and straw are crushed, mixed with animal manure and auxiliary materials for adjusting C / N ratio in proportion, and evenly mixed to obtain an organic mixture with C / N ratio of 25:1 to 30:1.

[0082] ②The fermentation liquor of Bacillus megaterium NT2 is evenly sprayed on the organic mixture in a mass ratio of 1-5%, and water is added to adjust the moisture content of the mixture to 50%-60%.

[0083] ④The material is stacked into a pile with a height of 1-1.5 meters and a width of 1.5-2 meters, and a straw mat is covered to ensure that the temperature is between 28-37℃ and the humidity is between 60-80%.

[0084] ⑤When the temperature of the pile rises above 65℃ and then starts to drop and stabilizes below 50℃, the pile is thoroughly turned over, and 1-1.5% of a mixture of nano-selenium and bacterial bodies is evenly added as a selenium source for continued fermentation.

[0085] ⑥Mature management and detection analysis: when the temperature of the pile drops to the ambient temperature and remains stable, the material is dark brown, loose, odorless and has a soil fragrance, indicating that the composting is complete. After the organic fertilizer composition and selenium content meet the standards, nano-selenium organic fertilizer is obtained.

[0086]

[0087] The above nano-selenium organic fertilizer is used in tea gardens. The control group is not treated, and the treatment groups are activated high-selenium rock powder organic fertilizer and nano-selenium organic fertilizer. Fertilization research is carried out in multiple sites in Ankang tea gardens. One bud and two leaf tea leaves are picked in 1.5 months, 3 months and 4.5 months for selenium content detection. See Table 4. The results show that before selenium application, the selenium content of tea leaves in the two test areas is lower than 0.10 mg / kg, which does not meet the standard of selenium-rich tea. After 1.5 months of selenium application, the selenium content of T1 (activated high-selenium rock powder organic fertilizer) treatment is 0.18-0.25 mg / kg, and the selenium content of T2 (nano-selenium organic fertilizer) treatment is 0.34-0.42 mg / kg. After 3 months of selenium application, the selenium content of T1 (activated high-selenium rock powder organic fertilizer) treatment is 0.44-0.49 mg / kg, and the selenium content of T2 (nano-selenium organic fertilizer) treatment is 0.69-0.76 mg / kg. After 4.5 months of selenium application, the selenium content of T1 (activated high-selenium rock powder organic fertilizer) treatment is 0.71-0.74 mg / kg, and the selenium content of T2 (nano-selenium organic fertilizer) treatment is 1.24-1.29 mg / kg. After selenium application, the selenium content of tea leaves increases from below the lower limit of the selenium content of selenium-rich tea to the standard of selenium-rich tea, and the selenium content of T2 (nano-selenium organic fertilizer) tea leaves increases by 55.10%-88.89% compared with that of T1 (activated high-selenium rock powder organic fertilizer) tea leaves. Therefore, nano-selenium reduced by Bacillus megaterium NT2 has higher selenium bioavailability and can effectively increase the selenium content of tea leaves.

[0088] The contents of Pb, Cd and As in tea were detected according to national standards GB 5009.12, GB 5009.15 and GB 5009.11. The contents of Pb, Cd and As in the control group were 0.28-0.44 mg / kg, 0.019-0.044 mg / kg and 0.055-0.092 mg / kg, respectively. The contents of Pb, Cd and As in tea treated with T2 (nano-selenium organic fertilizer) 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 by the Bacillus megaterium did not significantly increase the contents of Pb, Cd and As in tea. According to GB 2762-2022 "National Food Safety Standard Limits of Contaminants in Foods", the content of lead in tea should be ≤5.0 mg / kg. According to NY 659-2003 "Limits of Chromium, Cadmium, Mercury, Arsenic and Fluoride in Tea", the contents of cadmium and arsenic in tea should be ≤1 mg / kg and ≤2 mg / kg, respectively. After applying the nano-selenium organic fertilizer, the contents of Pb, Cd and As in tea did not exceed the standards.

[0089]

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 a mixture of nano-selenium and bacterial cells, 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 a mixture of nano-selenium and microbial cells, 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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