Method for preparing nano-selenium by using microbial bacteria and application of nano-selenium

Abstract

The invention provides a method for preparing nano-selenium by using microbial bacteria and application, and belongs to the technical field of nano-selenium preparation, the method comprises the following steps: preparing primary nano-selenium by using three different strains through fermentation; the preparation method comprises the following steps: mixing 50-55 parts by weight of 4-vinyl benzyl trimethyl ammonium 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 octyl mercaptan, and reacting at 80-90 DEG C under the protection of nitrogen for 7-9 hours to obtain a modifier; and modifying the primary nano-selenium by using a modifier to obtain the nano-selenium prepared by using the microbial bacteria. The primary nano-selenium prepared by the method disclosed by the invention is high in reduction efficiency, the synthesized primary nano-selenium is uniform in granularity, and the storage stability can be improved and the agglomeration problem can be improved by modifying the primary nano-selenium through the modifier.

Description

Technical Field

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

[0002] The application of nano-selenium in agriculture is an emerging research field. Due to its unique physical and chemical properties, nano-selenium has shown 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, and can enhance the ability of plants to resist various adversities, 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 the 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] The conversion rate and storage stability of nano-selenium currently prepared by microorganisms on the market are not ideal. Therefore, a method and application of preparing nano-selenium using microorganisms is urgently needed. 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) inoculating three strains on slant culture medium respectively, culturing and activating at 30-35° C., and obtaining three slant cultured bacterial strains after the colonies have grown all over the slant; the three strains are Bacillus numbered CCTCC AB 205273, Bacillus amyloliquefaciens numbered CCTCC AB 2013062, and Lactobacillus plantarum numbered SHBCC D16929;

[0009] (2) inoculating the three slant cultured bacteria into beef extract peptone liquid medium containing sodium selenite, respectively, and culturing 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) freezing and centrifuging the fermentation broth to collect the bacterial cells, washing them with sterile physiological saline for 3-4 times, suspending them with sterile distilled water, ultrasonically disrupting them, and collecting the supernatant containing nano-selenium after centrifugation. The supernatant was centrifuged to collect the precipitate, and the precipitate was washed with deionized water for 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 solution is inoculated into the fermentation medium at an inoculation rate of 1.6%-1.9%; the Bacillus amyloliquefaciens seed solution is inoculated into the fermentation medium at an inoculation rate of 1%-1.3%; and the Lactobacillus plantarum seed solution 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] There are many types of bacteria that can be prepared on the market, but the reduction efficiency of nano-selenium prepared using selenium salts is not high. The present invention can improve the reduction efficiency of nano-selenium prepared by microbial agents by fermenting three specific bacteria at the same time. These three bacteria have a synergistic effect with each other and can improve the conversion rate. It is also found that the three bacteria can improve the uniformity of nano-selenium particle size under specific addition conditions. Analysis shows that these three strains have different reduction abilities and growth characteristics, which can affect the formation and particle size of nano-selenium particles.

[0018] Furthermore, the 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 the 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] Further, 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, 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, and in order to ensure the reaction efficiency, the modification usually needs to be carried out in an organic solvent, generating a large amount of alcohol by-products, and the generation of three wastes is high, 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 to modify nano-selenium, and at the same time, the compatibility with the components in the fertilizer after modification is better, 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 nano-selenium to form a hydrophobic area, which is not easy to cause agglomeration due to moisture absorption. By finely regulating the hydrophobicity of the allyl polyethylene glycol ratio in the modifier, and then supplemented by dosage control, the storage stability under high temperature and variable temperature can be improved.

[0024] The present invention also provides 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 the synthesized primary nano-selenium has uniform particle size. Modification of the primary nano-selenium by a modifier can improve storage stability, improve agglomeration problems, and increase compatibility with organic components in fertilizers.

[0028] 2. The present invention can improve the reduction efficiency of microbial agents for preparing nano-selenium by fermenting three strains simultaneously. The three strains have a synergistic effect with each other and can improve the conversion rate. It is also found that the three strains can improve the uniformity of nano-selenium particle size under specific addition conditions.

[0029] 3. The present invention improves the storage stability of nano-selenium at room temperature by using a modifier to modify the nano-selenium. 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 of the allyl polyethylene glycol ratio in the modifier and then controls the dosage, thereby improving storage stability at variable temperatures and high temperatures and preventing aggregation from occurring easily. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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., No. 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, numbered SHBCC D16929, was purchased from Shanghai Microbiological Collection Center.

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

[0037] Pseudomonas aeruginosa, numbered 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 on slant culture media respectively, and static cultured and activated at 32° C., and three slant cultured bacterial strains were obtained when the colonies covered the slant; 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 bacteria were inoculated into beef extract peptone liquid medium containing sodium selenite, respectively, and cultured at 32° C. and 120 r / min for 18 h to obtain Bacillus seed liquid, Bacillus amyloliquefaciens seed liquid, and Lactobacillus plantarum seed liquid, respectively; the preparation method of 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 seed liquid into the fermentation medium at an inoculation rate of 1.7%; inoculating the Bacillus amyloliquefaciens seed liquid into the fermentation medium at an inoculation rate of 1.2%; and inoculating the Lactobacillus plantarum seed liquid into the fermentation medium at an inoculation rate of 0.6%; and performing aerobic fermentation under the following fermentation conditions: stirring rate of 120 r / min, temperature maintained at 32° C., and fermentation time of 33 h to obtain a fermentation liquid;

[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, pH=7.3;

[0046] (4) The fermentation liquid was frozen and centrifuged to collect the bacterial cells, which were washed with sterile physiological saline for 4 times, suspended with sterile distilled water, and ultrasonically disrupted at a power of 550 W, a frequency of 35 kHz, a start-stop interval of 12 s, and a disruption time of 35 min; after centrifugation, the supernatant containing nano-selenium was collected, the supernatant was centrifuged, the precipitate was collected, and the precipitate was washed with deionized water for 3 times 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 of them 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 on slant culture media respectively, and static cultured and activated at 30° C., and three slant cultured bacterial strains were obtained when the colonies covered the slant; the three strains were Bacillus with the number CCTCC AB 205273, Bacillus amyloliquefaciens with the number CCTCC AB 2013062, and Lactobacillus plantarum with the number SHBCC D16929;

[0052] (2) The three slant cultured bacterial species were inoculated into beef extract peptone liquid medium containing sodium selenite, respectively, and cultured at 35° C. and 100 r / min for 20 h to obtain Bacillus seed liquid, Bacillus amyloliquefaciens seed liquid, and Lactobacillus plantarum seed liquid, respectively; the preparation method of 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 inoculation rate of 1.9%; inoculating the Bacillus amyloliquefaciens seed liquid into the fermentation medium at an inoculation rate of 1%, and inoculating the Lactobacillus plantarum seed liquid into the fermentation medium at an inoculation rate of 0.7%, and performing aerobic fermentation, wherein the fermentation conditions are: a stirring rate of 100 r / min, a temperature maintained at 35° C., and a fermentation time of 30 h to obtain a fermentation liquid;

[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, pH=7.2;

[0055] (4) The fermentation liquid was frozen and centrifuged to collect the bacterial cells, which were washed three times with sterile physiological saline, suspended with sterile distilled water, and ultrasonically broken. The ultrasonic breaking power was 600 W, the frequency was 30 KHz, the start and stop interval was 15 s, and the breaking was 30 min; 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 inoculation rate of 1.2%; the Bacillus amyloliquefaciens seed liquid is inoculated into the fermentation medium at an inoculation rate of 1.2%, and the Lactobacillus plantarum seed liquid is inoculated into the fermentation medium at an inoculation rate 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 of CCTCC AB 2013062 is used for aerobic fermentation, and the seed liquid of Bacillus amyloliquefaciens is inoculated into the fermentation medium at an inoculation 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 the reaction 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 them are 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 which are purchased from Hai'an Petrochemical Plant, Jiangsu Province.

[0073] Performance Testing

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

[0075] Purity = the mass of selenium measured in primary nano-selenium / the 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 performed on the nano-selenium prepared using microorganisms in Examples 1-2 and Comparative Examples 1-6.

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

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

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

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

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

[0086] If there is no obvious change in the preparation of nano-selenium using microorganisms, it will be recorded as passed, and if there is agglomeration, it will be recorded as failed.

[0087] Table 2 Stability test results

[0088] Normal temperature appearance Cold resistant Hot and cold cycle 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 Not passed pass Not passed Not passed Comparative Example 5 pass pass Not passed Not passed Comparative Example 6 pass pass Not passed Not passed

[0089] As shown in Table 2, the nano-selenium prepared by microbial bacteria in Examples 1-2 has high stability. However, the comparative example is significantly worse than the embodiment in terms of corresponding performance tests because the necessary technical solutions are not adopted. In Comparative Example 5, the modification method is changed, and it can be seen that the storage stability effect is reduced. The modification scheme using silane European linking agent cannot make nano-selenium have a more ideal hydrophobicity and dispersibility. The composition scheme 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 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) inoculating three strains on slant culture medium respectively, culturing and activating at 30-35° C., and waiting for the colonies to grow all over the slant to obtain three slant cultured bacterial strains; the three strains are Bacillus numbered CCTCC AB 205273, Bacillus amyloliquefaciens numbered CCTCCAB 2013062, and Lactobacillus plantarum numbered SHBCC D16929; (2) inoculating the three slant cultured bacteria into beef extract peptone liquid medium containing sodium selenite, respectively, and culturing 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 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; (4) freezing and centrifuging the fermentation broth to collect the bacterial cells, washing them with sterile physiological saline for 3-4 times, suspending them with sterile distilled water, ultrasonically disrupting them, and collecting the supernatant containing nano-selenium after centrifugation. The supernatant was centrifuged to collect the precipitate, and the precipitate was washed with deionized water for 2-3 times to obtain primary nano-selenium; (5) mixing 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, reacting at 80-90° C. under nitrogen protection for 7-9 hours to obtain a modifier; (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.

2. The method for preparing nano-selenium using microorganisms according to claim 1, characterized in that: The method for preparing a 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, characterized in that: In the 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 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%.

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. Use of the method for preparing nano-selenium using microorganisms as described in any one of claims 1 to 8.

10. The use according to claim 9, characterized in that: The method for preparing nano-selenium using microorganisms is used in preparing fertilizers.