A method for preparing nano-selenium using microorganisms and its application

Abstract

The present invention provides a method and application for preparing nano-selenium using microorganisms, which belongs to the field of nano-selenium preparation technology. The method comprises the following steps: preparing primary nano-selenium using three different strains of bacteria fermentation; 50-55 parts by weight of 4-vinylbenzyltrimethylammonium chloride and 120-125 parts by weight of allyl polyethylene glycol, 1.1-1.3 parts by weight of benzoyl peroxide and 2.2-2.6 parts by weight of octanethiol are mixed, at 80-90 DEG C, under nitrogen protection, reacted for 7-9h to obtain a modifier; primary nano-selenium is modified using a modifier, and nano-selenium is prepared using microorganisms. The primary nano-selenium prepared by the method of the present invention has high reduction efficiency, and the synthesized primary nano-selenium has uniform particle size, and its modification by a modifier can improve storage stability and improve agglomeration and caking problems.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-selenium, and in particular relates to a method for preparing nano-selenium using microorganisms and its application. Background Art

[0002] The application of nano-selenium in agriculture is an emerging research area. Due to its unique physical and chemical properties, nano-selenium shows great potential in increasing crop yields, enhancing plant stress resistance, and improving the quality of agricultural products. Nano-selenium can be used as a fertilizer supplement to promote plant growth and development, thereby increasing crop yields. It can also enhance plants' ability to withstand various adverse conditions, such as drought, salinity, and heavy metal pollution.

[0003] The use of microorganisms (such as bacteria, fungi, etc.) to prepare nano-selenium is an environmentally friendly and promising method, but the following technical problems currently exist in this process: 1. Selenide conversion efficiency: The efficiency of microorganisms in converting selenide into nano-selenium particles may not be high, resulting in low yields. 2. Nanoparticle size and distribution: The size and distribution of nano-selenium particles are uneven, which may lead to unstable application effects. 3. Stability of nanoparticles: Nano-selenium particles may easily aggregate or lose stability after preparation. 4. Biosafety: The use of microorganisms to prepare nano-selenium may bring biosafety issues.

[0004] Currently, the conversion rate and storage stability of nano-selenium prepared by microorganisms on the market are not ideal. Therefore, there is an urgent need for a method and application of preparing nano-selenium using microorganisms. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing nano-selenium using microorganisms and its application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing nano-selenium using microorganisms, the method comprising the following steps:

[0008] (1) The three strains were inoculated onto slant culture media, and activated by static culture at 30-35° C., and the three slant cultured strains were obtained after the colonies had grown all over the slant; the three strains were Bacillus subtilis numbered CCTCC AB 205273, Bacillus amyloliquefaciens numbered CCTCC AB 2013062, and Lactobacillus plantarum numbered SHBCC D16929;

[0009] (2) The three slant cultured strains were inoculated into beef extract peptone liquid medium containing sodium selenite, and cultured at 30-35° C. and 100-150 rpm for 15-20 h to obtain Bacillus seed liquid, Bacillus amyloliquefaciens seed liquid, and Lactobacillus plantarum seed liquid, respectively;

[0010] (3) inoculating the Bacillus seed liquid, the Bacillus amyloliquefaciens seed liquid, and the Lactobacillus plantarum seed liquid into the same fermentation medium for aerobic fermentation to obtain a fermentation liquid;

[0011] (4) Refrigerate and centrifuge the fermentation broth to collect the bacterial cells, rinse them 3-4 times with sterile physiological saline, suspend them in sterile distilled water, and ultrasonically disrupt them. After centrifugation, collect the supernatant containing nano-selenium, centrifuge the supernatant, collect the precipitate, and wash the precipitate with deionized water 2-3 times to obtain primary nano-selenium;

[0012] (5) mixing 50-55 parts by weight of 4-vinylbenzyltrimethylammonium chloride (CAS: 26616-35-3), 120-125 parts by weight of allyl polyethylene glycol, 1.1-1.3 parts by weight of benzoyl peroxide, and 2.2-2.6 parts by weight of octanethiol, and reacting at 80-90° C. under nitrogen protection for 7-9 hours to obtain a modifier;

[0013] (6) Dispersing primary nano-selenium in water 10-15 times its weight, ultrasonically treating for 20-30 minutes, adding a modifier, stirring and reacting at 60-70° C. for 1.5-2 hours; centrifuging, drying, and obtaining nano-selenium prepared using microbial bacteria.

[0014] Slant culture medium was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: R22702 slant culture medium (containing agar).

[0015] Furthermore, the Bacillus seed liquid is inoculated into the fermentation medium at an inoculation rate of 1.6%-1.9%; the Bacillus amyloliquefaciens seed liquid is inoculated into the fermentation medium at an inoculation rate of 1%-1.3%; and the Lactobacillus plantarum seed liquid is inoculated into the fermentation medium at an inoculation rate of 0.4%-0.7%.

[0016] Furthermore, the fermentation conditions are: stirring rate 100-150r / min, temperature maintained at 30-35°C, and fermentation time 30-35h.

[0017] Currently, there are many strains available on the market that can be produced, but the reduction efficiency of nano-selenium produced using selenium salts is not high. The present invention improves the reduction efficiency of nano-selenium produced using microbial agents by simultaneously fermenting three specific strains to produce nano-selenium. These three strains have a synergistic effect with each other, which can increase the conversion rate. It was also found that the three strains, when added in specific amounts, can improve the uniformity of nano-selenium particle size. Analysis shows that these three strains have different reducing abilities and growth characteristics, which can affect the formation and particle size of nano-selenium particles.

[0018] Furthermore, a method for preparing a beef extract peptone liquid culture medium containing sodium selenite comprises the following steps: adding 200-300 mg of sodium selenite to 1 L of beef extract peptone liquid culture medium. The beef extract peptone liquid culture medium was purchased from Beijing Hongrun Baoshun Technology Co., Ltd.

[0019] Furthermore, the fermentation medium includes the following components: 8-9 g / L glucose, 6-7 g / L peptone, 2-3 g / L ammonium sulfate, 0.8-1.7 g / L magnesium sulfate heptahydrate, 0.2-0.9 g / L potassium dihydrogen phosphate, 0.7-1.6 g / L dipotassium hydrogen phosphate, 1-2 g / L sodium chloride and 2.5-3.7 g / L sodium selenite, pH = 7.2-7.4.

[0020] Furthermore, in step (4), the ultrasonic crushing power is 500W-600W, the frequency is 30-40KHz, the start-stop interval is 10-15s, and the crushing time is 30-40min.

[0021] Furthermore, the allyl polyethylene glycol is a mixture of allyl polyethylene glycol with a hydroxyl value of 74-88 mgKOH / g, allyl polyethylene glycol with a hydroxyl value of 53-63 mgKOH / g, and allyl polyethylene glycol with a hydroxyl value of 50-55 mgKOH / g in a weight ratio of 1: (1.2-1.4): (0.5-0.7), all of which are purchased from Hai'an Petrochemical Plant in Jiangsu Province.

[0022] Furthermore, the weight percentage of the modifier in the nano-selenium is 3-6%.

[0023] Nano-selenium is an important component in compound fertilizers, but nano-selenium will agglomerate when stored for a long time, which affects the quality of nano-selenium and leads to unsatisfactory fertilizer use effect. At present, the modification of nano-selenium in fertilizers is often modified by silane coupling agents. In order to ensure reaction efficiency, the modification usually needs to be carried out in an organic solvent, which generates a large amount of alcohol by-products and high levels of three wastes, which does not conform to the current trend of green development. The present invention improves the storage stability of nano-selenium at room temperature by using a modifier. At the same time, the modified nano-selenium has better compatibility with the components in the fertilizer, and the modification method is more environmentally friendly. The present invention adsorbs the modifier on nano-selenium, and the hydrophobic side chain of the modifier shields the surface space of the nano-selenium to form a hydrophobic area, which is not easy to agglomerate due to moisture absorption. By finely regulating the hydrophobicity of the allyl polyethylene glycol ratio in the modifier and then controlling the dosage, the storage stability under high temperature and variable temperature can be improved.

[0024] The present invention also provides an application of the method for preparing nano-selenium using microorganisms.

[0025] Furthermore, the method for preparing nano-selenium using microorganisms is used in preparing fertilizers.

[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0027] 1. The primary nano-selenium prepared by the method of the present invention has high reduction efficiency and uniform particle size. Modification of the primary nano-selenium by a modifier can improve storage stability, alleviate agglomeration problems, and increase compatibility with organic components in fertilizers.

[0028] 2. The present invention improves the reduction efficiency of nano-selenium produced by microbial inoculants by simultaneously fermenting three bacterial strains. These three bacterial strains exhibit synergistic effects, increasing conversion rates. Furthermore, it has been found that the addition of these three bacterial strains, when used in specific amounts, can improve the uniformity of nano-selenium particle size.

[0029] 3. The present invention improves the storage stability of nano-selenium at room temperature by modifying the nano-selenium using a modifier. At the same time, the modified nano-selenium has better compatibility with the components in the fertilizer, and the modification method is more environmentally friendly.

[0030] 4. The present invention finely controls the hydrophobicity by adjusting the proportion of allyl polyethylene glycol in the modifier and then controls the dosage, thereby improving storage stability at variable temperatures and high temperatures and preventing aggregation. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0032] The strains and other raw materials used in the present invention are all commercially available products:

[0033] Bacillus sp., accession number: CCTCC AB 205273, was purchased from China Center for Type Culture Collection.

[0034] Bacillus amyloliquefaciens, accession number: CCTCC AB 2013062, was purchased from China Center for Type Culture Collection.

[0035] Lactobacillus plantarum, number: SHBCC D16929, was purchased from Shanghai Microbiological Collection Center.

[0036] Bacillus subtilis (Bacillus subtilis), number: SHMCC D50742, was purchased from Shanghai Collection of Microorganisms.

[0037] Pseudomonas aeruginosa, number: SHMCC D71780, was purchased from Shanghai Microbiological Collection Center.

[0038] Slant culture medium was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: R22702 slant culture medium (containing agar).

[0039] Beef extract peptone liquid medium was purchased from Beijing Hongrun Baoshun Technology Co., Ltd.

[0040] Example 1

[0041] This embodiment provides a method for preparing nano-selenium using microorganisms, the method comprising the following steps:

[0042] (1) The three strains were inoculated onto slant culture media, and activated by static culture at 32°C. After the colonies covered the slant, three slant cultured strains were obtained; the three strains were Bacillus subtilis numbered CCTCC AB 205273, Bacillus amyloliquefaciens numbered CCTCC AB 2013062, and Lactobacillus plantarum numbered SHBCC D16929;

[0043] (2) The three slant cultured strains were inoculated into beef extract peptone liquid medium containing sodium selenite, and cultured at 32°C and 120 rpm for 18 h to obtain Bacillus seed liquid, Bacillus amyloliquefaciens seed liquid, and Lactobacillus plantarum seed liquid, respectively. The method for preparing the beef extract peptone liquid medium containing sodium selenite comprises the following steps: adding 250 mg of sodium selenite to 1 L of beef extract peptone liquid medium;

[0044] (3) inoculating the Bacillus subtilis seed solution into the fermentation medium at an inoculum size of 1.7%; inoculating the Bacillus amyloliquefaciens seed solution into the fermentation medium at an inoculum size of 1.2%; and inoculating the Lactobacillus plantarum seed solution into the fermentation medium at an inoculum size of 0.6%; and performing aerobic fermentation under the following fermentation conditions: a stirring rate of 120 r / min, a temperature maintained at 32° C., and a fermentation time of 33 h to obtain a fermentation broth;

[0045] The fermentation medium comprises the following components: 8.6 g / L glucose, 6.3 g / L peptone, 2.2 g / L ammonium sulfate, 1 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium dihydrogen phosphate, 1.1 g / L dipotassium hydrogen phosphate, 1.5 g / L sodium chloride and 3 g / L sodium selenite, with a pH of 7.3;

[0046] (4) The fermentation broth was frozen and centrifuged to collect the bacterial cells, which were washed four times with sterile physiological saline, suspended in sterile distilled water, and ultrasonically disrupted at a power of 550 W, a frequency of 35 kHz, a start-stop interval of 12 seconds, and a disruption time of 35 minutes; after centrifugation, the supernatant containing nano-selenium was collected, the supernatant was centrifuged, the precipitate was collected, and the precipitate was washed three times with deionized water to obtain primary nano-selenium;

[0047] (5) 52 parts by weight of 4-vinylbenzyltrimethylammonium chloride (CAS: 26616-35-3) and 122 parts by weight of allyl polyethylene glycol, 1.2 parts by weight of benzoyl peroxide and 2.4 parts by weight of octanethiol were mixed and reacted at 85°C under nitrogen protection for 8 hours to obtain a modifier; the allyl polyethylene glycol was a mixture of allyl polyethylene glycol with a hydroxyl value of 74-88 mgKOH / g, allyl polyethylene glycol with a hydroxyl value of 53-63 mgKOH / g and allyl polyethylene glycol with a hydroxyl value of 50-55 mgKOH / g in a weight ratio of 1:1.3:0.6; all were purchased from Hai'an Petrochemical Plant, Jiangsu Province.

[0048] (6) Dispersing primary nano-selenium in water 12 times its weight, ultrasonically treating for 25 minutes, adding a modifier, wherein the modifier accounts for 5% of the weight of the nano-selenium; stirring the reaction at 65° C. for 1.8 hours; centrifuging and drying to obtain nano-selenium prepared using microbial bacteria.

[0049] Example 2

[0050] This embodiment provides a method for preparing nano-selenium using microorganisms, the method comprising the following steps:

[0051] (1) The three strains were inoculated onto slant culture media, and activated by static culture at 30°C until the colonies covered the slant, thereby obtaining three slant cultured bacterial strains; the three strains were Bacillus subtilis numbered CCTCC AB 205273, Bacillus amyloliquefaciens numbered CCTCC AB 2013062, and Lactobacillus plantarum numbered SHBCC D16929;

[0052] (2) The three slant cultured strains were inoculated into beef extract peptone liquid medium containing sodium selenite, respectively, and cultured at 35°C and 100 rpm for 20 h to obtain Bacillus seed liquid, Bacillus amyloliquefaciens seed liquid, and Lactobacillus plantarum seed liquid, respectively. The method for preparing the beef extract peptone liquid medium containing sodium selenite comprises the following steps: adding 200 mg of sodium selenite to 1 L of beef extract peptone liquid medium;

[0053] (3) inoculating the Bacillus seed liquid into the fermentation medium at an inoculum size of 1.9%; inoculating the Bacillus amyloliquefaciens seed liquid into the fermentation medium at an inoculum size of 1%, and inoculating the Lactobacillus plantarum seed liquid into the fermentation medium at an inoculum size of 0.7%, and performing aerobic fermentation under the following fermentation conditions: a stirring rate of 100 r / min, a temperature maintained at 35° C., and a fermentation time of 30 h to obtain a fermentation broth;

[0054] The fermentation medium comprises the following components: 9 g / L glucose, 6 g / L peptone, 3 g / L ammonium sulfate, 0.8 g / L magnesium sulfate heptahydrate, 0.9 g / L potassium dihydrogen phosphate, 1.6 g / L dipotassium hydrogen phosphate, 1 g / L sodium chloride and 3.7 g / L sodium selenite, with a pH of 7.2;

[0055] (4) The fermentation broth was frozen and centrifuged to collect the bacterial cells, which were rinsed three times with sterile saline, suspended in sterile distilled water, and ultrasonically disrupted at a power of 600 W, a frequency of 30 kHz, a start-stop interval of 15 seconds, and a disruption time of 30 minutes; after centrifugation, the supernatant containing nano-selenium was collected, the supernatant was centrifuged, the precipitate was collected, and the precipitate was washed twice with deionized water to obtain primary nano-selenium;

[0056] (5) 50 parts by weight of 4-vinylbenzyltrimethylammonium chloride (CAS: 26616-35-3) and 125 parts by weight of allyl polyethylene glycol, 1.1 parts by weight of benzoyl peroxide and 2.6 parts by weight of octanethiol were mixed and reacted at 80°C under nitrogen protection for 9 hours to obtain a modifier; the allyl polyethylene glycol was a mixture of allyl polyethylene glycol with a hydroxyl value of 74-88 mgKOH / g, allyl polyethylene glycol with a hydroxyl value of 53-63 mgKOH / g and allyl polyethylene glycol with a hydroxyl value of 50-55 mgKOH / g in a weight ratio of 1:1.2:0.5. All of them were purchased from Hai'an Petrochemical Plant, Jiangsu Province.

[0057] (6) Dispersing primary nano-selenium in water 15 times its weight, ultrasonically treating for 20 minutes, adding a modifier, wherein the modifier accounts for 6% of the weight of the nano-selenium; stirring the reaction at 60° C. for 2 hours; centrifuging and drying to obtain nano-selenium prepared using microbial bacteria.

[0058] Comparative Example 1

[0059] The difference between this comparative example and Example 1 is that the Bacillus seed liquid is inoculated into the fermentation medium at an inoculum size of 1.2%; the Bacillus amyloliquefaciens seed liquid is inoculated into the fermentation medium at an inoculum size of 1.2%, and the Lactobacillus plantarum seed liquid is inoculated into the fermentation medium at an inoculum size of 1.2% for aerobic fermentation.

[0060] Comparative Example 2

[0061] The difference between this comparative example and Example 1 is that only Bacillus amyloliquefaciens with the number CCTCC AB 2013062 was used for aerobic fermentation, and the seed liquid of Bacillus amyloliquefaciens was inoculated into the fermentation medium at an inoculum rate of 3.6%.

[0062] Comparative Example 3

[0063] The difference between this comparative example and Example 1 is:

[0064] The three strains were Bacillus amyloliquefaciens with the number CCTCC AB 2013062, Bacillus subtilis with the number SHMCC D50742, and Pseudomonas aeruginosa with the number SHMCC D71780;

[0065] The Bacillus subtilis seed solution was inoculated into the fermentation medium at an inoculation rate of 1.7%; the Bacillus amyloliquefaciens seed solution was inoculated into the fermentation medium at an inoculation rate of 1.2%; and the Pseudomonas aeruginosa seed solution was inoculated into the fermentation medium at an inoculation rate of 0.6% for aerobic fermentation.

[0066] Comparative Example 4

[0067] The difference between this comparative example and Example 1 is that a silane coupling agent is used for modification.

[0068] The specific steps are: dispersing primary nano-selenium in water 15 times its weight, ultrasonically treating for 20 minutes, adding silane coupling agent KH550, wherein the silane coupling agent KH550 accounts for 6% of the weight of the nano-selenium; stirring and reacting at 60°C for 2 hours; centrifuging and drying to obtain nano-selenium prepared using microbial bacteria.

[0069] Comparative Example 5

[0070] The difference between this comparative example and Example 1 is that the allyl polyethylene glycol is a mixture of allyl polyethylene glycol with a hydroxyl value of 74-88 mgKOH / g, allyl polyethylene glycol with a hydroxyl value of 53-63 mgKOH / g, and allyl polyethylene glycol with a hydroxyl value of 50-55 mgKOH / g in a weight ratio of 1:1:1. All of the ingredients were purchased from Hai'an Petrochemical Plant in Jiangsu Province.

[0071] Comparative Example 6

[0072] The difference between this comparative example and Example 1 is that the allyl polyethylene glycol is a mixture of allyl polyethylene glycol with a hydroxyl value of 66-77 mgKOH / g, allyl polyethylene glycol with a hydroxyl value of 27-30 mgKOH / g, and allyl polyethylene glycol with a hydroxyl value of 21-24 mgKOH / g in a weight ratio of 1:1.2:0.5. All of the ingredients were purchased from Hai'an Petrochemical Plant in Jiangsu Province.

[0073] Performance Testing

[0074] 1. The performance tests of the primary nano-selenium prepared in Examples 1-2 and Comparative Examples 1-3 were carried out in accordance with the national standard GB5009.93-2017. The results are shown in Table 1.

[0075] Purity = mass of selenium measured in primary nano-selenium / mass of primary nano-selenium * 100%.

[0076] Conversion rate = mass of selenium measured in primary nano-selenium / mass of selenium element in sodium selenite used in the whole method * 100%.

[0077] Table 1 Performance test results

[0078] Conversion Rate % purity% Average particle size nm Example 1 94.1 92.2 141±10 Example 2 93.4 92.1 145±12 Comparative Example 1 91.5 91.8 162±25 Comparative Example 2 90.9 91.4 176±36 Comparative Example 3 91.2 91.6 170±30

[0079] It can be seen from Table 1 that the primary nano-selenium prepared in Example 1-2 has a higher conversion rate, higher purity, and more uniform particle size. The conversion rate, purity, and particle size uniformity of the primary nano-selenium obtained by aerobic fermentation of a single strain of Bacillus amyloliquefaciens are poor. When different strains are used for co-fermentation with Bacillus amyloliquefaciens, the conversion rate and particle size uniformity decrease significantly. At the same time, changing the addition amount of the three strains will also affect the quality of the primary nano-selenium.

[0080] 2. Performance tests were conducted on the nano-selenium prepared using microorganisms prepared in Examples 1-2 and Comparative Examples 1-6.

[0081] Nano-selenium prepared using microorganisms was placed in the following four environments for observation to test the stability of the materials of the examples and comparative examples. The results are shown in Table 2.

[0082] Normal temperature: The sample is left at 25°C, 60% RH (relative humidity) for 90 days.

[0083] Cold resistance: -10℃ for 30 days.

[0084] Hot and cold cycles: -10°C, 25% RH; 25°C, 60% RH; 38°C, 75% RH, 24 hours at each temperature, 10 cycles in total.

[0085] High temperature acceleration: Test conditions are 50℃±1℃, 25%RH±5%RH for 30 days.

[0086] When using microorganisms to prepare nano-selenium, if there is no obvious change, it is recorded as passed; if there is agglomeration, it is recorded as failed.

[0087] Table 2 Stability test results

[0088] Normal temperature appearance Cold-resistant Hot and cold cycles high temperature Example 1 pass pass pass pass Example 2 pass pass pass pass Comparative Example 1 pass pass pass pass Comparative Example 2 pass pass pass pass Comparative Example 3 pass pass pass pass Comparative Example 4 Fail pass Fail Fail Comparative Example 5 pass pass Fail Fail Comparative Example 6 pass pass Fail Fail

[0089] As shown in Table 2, the nano-selenium prepared using microbial bacteria in Example 1-2 has high stability. However, the comparative example is significantly worse than the embodiment in terms of corresponding performance tests because it does not adopt the necessary technical solutions. In Comparative Example 5, the modification method is changed, and it can be seen that the storage stability effect is reduced. The modification solution using silane coupling agent cannot make nano-selenium have ideal hydrophobicity and dispersibility. The composition solution of allyl polyethylene glycol is not used in Comparative Examples 5-6. From the results, it can be seen that the storage stability at high temperature and variable temperature is affected. The above experimental results further prove the importance of the technical solution defined in the present invention for its technical effect.

[0090] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing nano-selenium using microorganisms, characterized in that: The method comprises the following steps: (1) The three strains were inoculated onto slant culture media, and activated by static culture at 30-35°C. The three slant cultured strains were obtained after the colonies had grown all over the slant. The three strains were Bacillus subtilis numbered CCTCC AB 205273, Bacillus amyloliquefaciens numbered CCTCCAB 2013062, and Lactobacillus plantarum numbered SHBCC D16929. (2) The three slant cultured strains were inoculated into beef extract peptone liquid medium containing sodium selenite, and cultured at 30-35°C and 100-150 rpm for 15-20 h to obtain Bacillus seed liquid, Bacillus amyloliquefaciens seed liquid, and Lactobacillus plantarum seed liquid, respectively; (3) inoculating the Bacillus subtilis seed liquid, the Bacillus amyloliquefaciens seed liquid, and the Lactobacillus plantarum seed liquid into the same fermentation medium, performing aerobic fermentation to obtain a fermentation liquid; (4) The fermentation liquid was frozen and centrifuged to collect the bacterial cells, which were washed 3-4 times with sterile physiological saline, suspended with sterile distilled water, ultrasonically disrupted, and centrifuged to collect the supernatant containing nano-selenium. The supernatant was centrifuged to collect the precipitate, and the precipitate was washed with deionized water 2-3 times to obtain primary nano-selenium; (5) Mix 50-55 parts by weight of 4-vinylbenzyltrimethylammonium chloride, 120-125 parts by weight of allyl polyethylene glycol, 1.1-1.3 parts by weight of benzoyl peroxide, and 2.2-2.6 parts by weight of octanethiol, and react at 80-90° C. under nitrogen protection for 7-9 hours to obtain a modifier; (6) Disperse the primary nano-selenium in water 10-15 times its weight, ultrasonically treat for 20-30 minutes, add a modifier, stir and react at 60-70°C for 1.5-2 hours; centrifuge and dry to obtain nano-selenium.

2. The method for preparing nano-selenium using microorganisms according to claim 1, wherein: The preparation method of beef extract peptone liquid culture medium containing sodium selenite comprises the following steps: adding 200-300 mg of sodium selenite into 1 L of beef extract peptone liquid culture medium.

3. The method for preparing nano-selenium using microorganisms according to claim 1, characterized in that: The fermentation medium comprises the following components: 8-9 g / L glucose, 6-7 g / L peptone, 2-3 g / L ammonium sulfate, 0.8-1.7 g / L magnesium sulfate heptahydrate, 0.2-0.9 g / L potassium dihydrogen phosphate, 0.7-1.6 g / L dipotassium hydrogen phosphate, 1-2 g / L sodium chloride and 2.5-3.7 g / L sodium selenite, with a pH of 7.2-7.

4.

4. The method for preparing nano-selenium using microorganisms according to claim 1, wherein: In step (4), the ultrasonic crushing power is 500W-600W, the frequency is 30-40KHz, the start-stop interval is 10-15s, and the crushing time is 30-40min.

5. The method for preparing nano-selenium using microorganisms according to claim 1, characterized in that: The allyl polyethylene glycol is a mixture of allyl polyethylene glycol with a hydroxyl value of 74-88 mgKOH / g, allyl polyethylene glycol with a hydroxyl value of 53-63 mgKOH / g and allyl polyethylene glycol with a hydroxyl value of 50-55 mgKOH / g in a weight ratio of 1: (1.2-1.4): (0.5-0.7).

6. The method for preparing nano-selenium using microorganisms according to claim 1, characterized in that: The weight percentage of the modifier to the nano-selenium is 3-6%.

7. The method for preparing nano-selenium using microorganisms according to claim 1, characterized in that: The Bacillus seed solution is inoculated into the fermentation medium at an inoculum rate of 1.6%-1.9%; The Bacillus amyloliquefaciens seed solution is inoculated into the fermentation medium at an inoculum rate of 1%-1.3%; The Lactobacillus plantarum seed liquid is inoculated into the fermentation medium at an inoculum rate of 0.4%-0.7%.

8. The method for preparing nano-selenium using microorganisms according to claim 1, characterized in that: The fermentation conditions are as follows: stirring rate 100-150 r / min, temperature maintained at 30-35° C., and fermentation time 30-35 h.

9. An application of the method for preparing nano-selenium using microorganisms according to any one of claims 1 to 8, characterized in that: Used in the preparation of fertilizers.