Method for green synthesis of zinc oxide nanosheets from aerospace mutant strain PS04-17

Abstract

The invention discloses a method for green synthesis of a zinc oxide nanosheet from an aerospace mutant strain PS04-17. The invention provides a new application of a space mutant strain PS04-17 in green synthesis of zinc oxide nanosheets, research shows that the PS04-17 strain has efficient capability of converting zinc nitrate hexahydrate into zinc oxide nanosheets, the conversion efficiency is high, the generated zinc oxide nanosheets are flat and smooth in surface and clear in edge, the sheet structure is presented, and the zinc oxide nanosheets have good application prospects. And orderly vertical or staggered folding is realized. The zinc oxide nanosheets are synthesized through the aerospace mutant strain PS04-17, more possibilities are provided for a green and innovative synthesis method, more feasible schemes are provided for synthesis of nano-zinc oxide through a microbiological method, and the synthesis method is efficient, simple, convenient, safe, low in cost, environmentally friendly and suitable for industrial production. The prepared zinc oxide nanosheet can be better applied to the fields of agriculture, industry, medical treatment and the like.

Description

Technical Field

[0001] This invention relates to the field of microbial synthesis of nanomaterials, and more specifically, to a method for the green synthesis of zinc oxide nanosheets from the space-bred mutant strain △PS04-17. Background Technology

[0002] Zinc oxide nanoparticles, as a novel metallic nanomaterial, have broad application prospects. Currently, nanomaterials are mainly synthesized through traditional physical and chemical methods, such as mechanical ball milling, hydrothermal methods, chemical vapor deposition, and thermal stirring, and are applied in various fields such as optoelectronic devices, medical antibacterial agents, environmental remediation, and new energy. The production of zinc oxide nanosheets using biological raw materials is a novel technology. Green plants contain polyphenols and proteins, which can replace chemical agents as reducing agents to generate zinc oxide nanoparticles. Existing research shows that Azolla oblongifolia contains abundant flavonoids and terpenes, which can be used as stabilizers in the nanoparticle synthesis process and to reduce the size of nanoparticles. However, the synthesis method using plants is cumbersome and inefficient.

[0003] Microbial synthesis of zinc oxide nanosheets is a green and sustainable method for preparing nanomaterials. Currently, published research primarily utilizes the metabolic activities of specific microorganisms (such as reduction, secretion of biological templates, or regulation of crystal growth). For example, bacteria, fungi, actinomycetes, and algae can reduce or oxidize zinc oxide nanosheets. For instance, Buszewski et al. utilized *Lactobacillus paracasei* (… Lactobacillus paracasei The addition of 3 mmol / L zinc nitrate and incubation at 26°C for 10 days confirmed the synthesis of ZnO-NPs by the appearance of white clusters at the bottom of the flask. Biosynthesis of zinc oxide nanosheets offers advantages such as simplicity, environmental friendliness, cost-effectiveness, and alignment with sustainable development goals. However, there are currently few reports on the microbial production of zinc oxide nanosheets, and the available microbial strains for synthesis are limited. This is because high concentrations of zinc ions in culture media can be lethal to microorganisms. Therefore, screening or breeding microbial strains with high tolerance to zinc ion stress is crucial for the green synthesis of zinc oxide nanosheets. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing microbial materials for synthesizing zinc nanosheets, and to provide a method for synthesizing zinc oxide nanosheets by a space mutant strain △PS04-17. The first objective of this invention is to provide the application of the aerospace mutant strain △PS04-17 in the preparation of zinc oxide nanosheets.

[0005] A second objective of this invention is to provide a method for biosynthesizing zinc oxide nanosheets.

[0006] A third object of the present application is to provide a zinc oxide nanosheet.

[0007] A fourth object of the present application is to provide a product.

[0008] The above objects of the present application are achieved by the following technical solutions. The present application provides a new application of the aerospace mutant strain △PS04-17 strain in the green synthesis of zinc oxide nanosheets. Research shows that the △PS04-17 strain has a high conversion efficiency of converting zinc nitrate hexahydrate into zinc oxide nanosheets, and the generated zinc oxide nanosheets have a smooth surface, clear edges, and exhibit a sheet structure with orderly vertical or staggered folding. The synthesis of zinc oxide nanosheets by the aerospace mutant strain △PS04-17 strain provides more possibilities for green and innovative synthesis methods, and provides more feasible solutions for the synthesis of zinc oxide nanomaterials by microbial methods. The synthesis method is efficient, simple, convenient, safe, low-cost, and environmentally friendly, and the prepared zinc oxide nanosheets can be better applied in the fields of agriculture, industry, and medicine.

[0009] Therefore, the present application provides the application of the aerospace mutant strain △PS04-17 of the Paenibacillus campinasensis-like bacteria Paenibacillus kribbensis in the preparation of zinc oxide nanosheets.

[0010] Further, the aerospace mutant strain △PS04-17 of the Paenibacillus campinasensis-like bacteria has been preserved in the Guangdong Microbial Culture Collection Center on March 17, 2022, and the preservation number is GDMCC NO:62233.

[0011] The present application provides a method for biosynthesizing zinc oxide nanosheets. After the aerospace mutant strain △PS04-17 of the Paenibacillus campinasensis-like bacteria is activated, it is inoculated into a fermentation medium containing zinc nitrate hexahydrate for fermentation culture. The fermentation broth is centrifuged, the precipitate is collected, washed, centrifuged again, purified, and freeze-dried to obtain

[0012] Preferably, the seed liquid of the △PS04-17 strain is inoculated into the fermentation medium at an inoculation amount of 2-5%.

[0013] Preferably, the fermentation medium uses a modified Czapek culture medium, and the formula is: zinc nitrate hexahydrate 1-5 g / L, sucrose 25-30 g / L, potassium phosphate dibasic 1-1.3 g / L, magnesium sulfate heptahydrate 1-1.2 g / L, potassium chloride 0.5-0.8 g / L, ferrous sulfate heptahydrate 0.01-0.03 g / L, and pH 6.5-7.5.

[0014] More preferably, the medium formula of the improved Czapek medium is: sucrose 25~30 g / L, potassium phosphate dibasic 1~1.3 g / L, magnesium sulfate heptahydrate 1 g / L, potassium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, pH 6.5~7.5.

[0015] More preferably, the concentration of the zinc nitrate hexahydrate is 1.5~4.5 g / L.

[0016] Preferably, the fermentation culture conditions are: temperature 25~28℃, rotation speed 100~200 rpm or static culture, time 7~10 days.

[0017] Preferably, the concentration of the seed liquid of the strain is not less than 1×10 6 cuf / mL.

[0018] Preferably, the purification method is: first ultrasonic crushing, then extraction; the ultrasonic condition is set as power 400~500W, ultrasonic operation 3~5s, pause 3~5s, total time 30~40min.

[0019] More preferably, the purification method is: washing the precipitate with sterile water for 2~3 times, the ultrasonic condition is set as power 450W, ultrasonic operation 5s pause 5s, total operation time 30~40min, then differential centrifugation, extraction and freeze-drying to obtain pure zinc oxide nanosheets.

[0020] The application provides a zinc oxide nanosheet prepared by the above method.

[0021] The application also provides a product containing the above zinc oxide nanosheet.

[0022] The application has the following beneficial effects: The application provides a new application of the aerospace mutant strain △PS04-17 in green synthesis of zinc oxide nanosheets. Research shows that the △PS04-17 strain has the ability to efficiently convert zinc nitrate hexahydrate into zinc oxide nanosheets, and has high conversion efficiency, which provides more feasible schemes for the microbial synthesis of zinc oxide nanosheet materials, and has the following advantages: (1) The application provides a new, green synthesis scheme of metal nanoparticles; (2) The method for preparing zinc oxide nanosheets provided by the application is efficient, simple, fast, low-cost and environmentally friendly; (3) The zinc oxide nanosheets synthesized by the aerospace mutant strain △PS04-17 of the genus Paenibacillus cereus have smooth surfaces, clear edges, present a sheet structure, and are orderly vertically or staggered folded, and the thickness range is 25~55 nm, which can be better applied to the fields of agriculture, industry, medical treatment and the like, and has good application value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The fermentation state of the △PS04-17 strain in the culture medium containing zinc nitrate hexahydrate (left in the figure is ck without adding seed liquid; right is the treatment with added seed liquid).

[0024] Figure 2 The effect diagram of the △PS04-17 strain fermented in the culture medium containing different concentrations of zinc nitrate hexahydrate for 7 days (from left to right in the figure are CK, 1.5 g / L zinc nitrate hexahydrate, 3.0 g / L zinc nitrate hexahydrate, and 4.5 g / L zinc nitrate hexahydrate).

[0025] Figure 3 The SEM scanning electron microscope image of zinc oxide nanosheets (A in the figure is the zinc oxide nanosheet without purification; B is the purified zinc oxide nanosheet).

[0026] Figure 4 The SEM scanning electron microscope image of pure zinc oxide nanosheets (A in the figure is the thickness marked; B is the width and length marked).

[0027] Figure 5 The SEM-EDS image of zinc oxide nanosheets (A in the figure is the SEM image and element analysis image of zinc oxide nanosheets; B is the distribution diagram of C, Pt, Zn, and O elements in the SEM of zinc oxide nanosheets in A). DETAILED DESCRIPTION

[0028] The present application will be further described below in conjunction with the drawings and specific examples of the specification, but the examples do not make any form of limitation to the present application. Unless otherwise specified, the reagents, methods, and equipment used in the present application are conventional reagents, methods, and equipment in the technical field.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] The Paenibacillus kribbensis used in the examples is a space mutant strain △PS04-17. Paenibacillus kribbensis The space mutant strain △PS04-17 of Paenibacillus kribbensis is the research result of the present application research group, and the existing research is recorded to be preserved in the Guangdong Microbial Culture Collection Center on March 17, 2022, with the preservation number of GDMCC NO:62233, the preservation address of Guangzhou Xianlie Middle Road No. 100, and the existing recorded Chinese patent publication number of CN114480222A.

[0031] Example 1 Determination of the conversion effect of strain △PS04-17 on zinc nitrate hexahydrate After the space mutant strain △PS04-17 of Paenibacillus kribbensis was activated, a culture medium with a concentration of 1×10 6The seed liquid of cuf / mL was inoculated into the modified Czapek culture solution containing 1.5 g / L zinc nitrate hexahydrate (the formula is: sucrose 25~30 g / L, potassium dihydrogen phosphate 1~1.3 g / L, magnesium sulfate heptahydrate 1~1.2 g / L, potassium chloride 0.5~0.8 g / L, ferrous sulfate heptahydrate 0.01~0.03 g / L, pH 6.5~7.5) at an inoculation amount of 3%, and the Czapek culture solution without seed liquid was used as a control treatment. The culture solution was placed at a temperature of 25~28°C for 10 days, and the state of the culture solution was observed to determine whether any changes occurred. Each treatment was repeated 3 times, and the liquid volume in each bottle was 500 mL.

[0032] The results of the determination are shown in Table 1. Figure 1 As shown in Table 1, after being placed for 10 days, white granular substances were produced in the culture solution with seed liquid, and some of the substances aggregated, indicating that the strain △PS04-17 had the ability to convert zinc nitrate hexahydrate into new substances.

[0033] Example 2: Determination of the tolerance of strain △PS04-17 to zinc nitrate hexahydrate and its conversion ability The modified Czapek culture solution containing different concentrations of 1.5~4.5 g / L zinc nitrate hexahydrate was prepared, and the Czapek culture solution containing 1.5 g / L zinc nitrate hexahydrate without seed liquid was used as a control treatment. The treatment group was the addition of 3% strain △PS04-17 seed liquid, and the concentration of zinc nitrate hexahydrate was 1.5 g / L, 3 g / L, and 4.5 g / L, respectively. The culture was carried out at a temperature of 25~28°C and a rotation speed of 200 rpm for 7 days. Each treatment was repeated 3 times, and the liquid volume in each bottle was 150 mL. The state of the culture solution after the addition of seed liquid was observed. Then, the fermentation broth of each treatment was centrifuged at 10000 rpm for 8 min, the supernatant was taken, and the zinc particle content was determined by flame atomic absorption spectrophotometry. The conversion rate of zinc nitrate hexahydrate was calculated according to the following formula: Conversion rate (%) = (C1-C2) / C1 x 100%; In the formula, C1 is the concentration of zinc in the original culture solution, and C2 is the concentration of zinc in the supernatant.

[0034] Subsequently, the white precipitate containing bacterial cells and zinc oxide nanosheets after centrifugation was disrupted by sonication in an ice bath. The sonication conditions were set to 450W power, with a 5-second run followed by a 5-second pause, for a total run time of 30-40 minutes. After sonication, the precipitate was centrifuged at 12000 rpm for 10 minutes. The precipitate was washed three times consecutively with 1.5 mol / L Tris-HCl buffer (pH 8.3). The resulting zinc oxide nanosheet precipitate was resuspended in ultrapure water, and n-octanol was added. The mixture was shaken for 5 minutes, centrifuged at 3000 rpm for 5 minutes, and then placed in a 4°C freezer for 24 hours to allow for clear phase separation. Once the zinc oxide nanosheets precipitated to the bottom of the centrifuge tube, the cells between the two phases were discarded, and the precipitate was washed twice with sterile water and dried in a supercritical dryer. After approximately one hour of drying, pure zinc oxide nanosheets were obtained. The yield of zinc oxide nanosheets for each treatment was calculated by weighing.

[0035] After culturing for 7 days using the above method, observe the results. Figure 2 As shown, except for the control group, bubbles were generated on the fermentation surface of the culture medium in all other treatments, and the solution became turbid. This indicates that the Cribben Bacillus space mutant strain △PS04-17 survived under all three treatment conditions, and white granular substances were generated at the bottom of all treatments, with some exhibiting aggregation.

[0036] Zn was calculated by measuring the zinc particle content. 2+ The conversion rates were shown in Table 1. The results showed that strain △PS04-17 could tolerate 4.5 g / L zinc nitrate hexahydrate, and its conversion rate decreased significantly with increasing zinc nitrate hexahydrate concentration in the culture medium. In the 4.5 g / L zinc nitrate hexahydrate medium, the conversion rate still reached 48.7%, with a zinc oxide nanosheet yield of 0.55 g, indicating a good conversion effect. Among the strains, △PS04-17 showed the highest conversion rate of 83.28% for 1.5 g / L zinc nitrate hexahydrate. Table 1. Results of conversion rate and yield determination of zinc nitrate hexahydrate.

[0037] Example 3: Preparation and Identification of Pure Zinc Oxide Nanosheets The fermentation broth obtained after the end of Example 2 was centrifuged at 10000 rpm for 8 min, the precipitate was collected and washed twice with sterile water, and then washed twice with 0.9% NaCl solution, sterile water was added to adjust the volume of the precipitated cells to not less than 1 / 2 of the original sample volume, and then the cells were broken by ice-bath ultrasonic treatment, with the ultrasonic conditions set as follows: power 450 W, ultrasonic operation 5 s pause 5 s, total operation time 30-40 min. After the ultrasonic treatment, the mixture was centrifuged at 12000 rpm for 10 min, and the precipitate was washed with 1.5 mol / L Tris-HCl buffer (pH 8.3) for 3 times, and then centrifuged to obtain the zinc oxide nanosheet precipitate of the culture.

[0038] The precipitate was resuspended in 4 mL ultrapure water, 2 mL n-octanol was added, and the mixture was shaken for 5 min, centrifuged at 3000 rpm for 5 min, and then placed in a 4°C refrigerator for 24 h to allow the solution to separate into two layers. The zinc oxide nanosheet precipitate was at the bottom of the centrifuge tube, and the cell fragments between the two layers were discarded. The precipitate was washed twice with sterile water, and finally the zinc oxide nanosheet was resuspended in 10 mL ultrapure water, centrifuged at 10000 rpm for 8 min, washed twice with PBS buffer with pH=7.5 for 10-15 min each time, fixed in 2.5% glutaraldehyde solution at 4°C overnight, washed with sterile water for 2-3 times for about 15 min each time, and then dried in a supercritical drying machine after centrifugation. The drying was performed for about 1 h to obtain pure zinc oxide nanosheets. At the same time, the white granular precipitate containing bacteria and pure zinc oxide nanosheets without ultrasonic purification treatment was used as a control group.

[0039] The sample prepared above was fixed on a sample stage, sprayed with pt, and tested. Imaging and photographing were performed using a Hitachi SU8010 scanning electron microscope, and EDS analysis of the elemental composition of the zinc oxide nanosheets was performed by SEM scanning electron microscope observation.

[0040] The SEM results of the precipitate containing bacteria and zinc oxide nanosheets before purification are shown in Figure 3 A, the precipitate without ultrasonic purification, the zinc oxide nanosheets and rod-shaped bacteria are inlaid distribution, and the size of the zinc oxide nanosheets is not uniform. After purification, the bacteria in the zinc oxide nanosheet precipitate are completely broken, and the surface of the zinc oxide nanosheet is smooth and flat, with clear edges, showing a sheet structure, and orderly vertical or staggered folding, as shown in Figure 3 B and Figure 4 , and the thickness thereof is in the range of 25-55 nm.

[0041] The SEM-EDS of the zinc oxide nanosheets is shown in Figure 5As shown, A is an SEM image and an element analysis image of the zinc oxide nanosheet, and the element analysis result shows characteristic peaks of C, O, Zn and Pt; B is a distribution diagram of C, Pt, Zn and O elements in the SEM of the zinc oxide nanosheet in A, respectively. The distribution of C, Pt, Zn and O elements in the zinc oxide nanosheet can be seen that the zinc oxide nanosheet converges in the central part, the C element is not concentrated, the Pt element is scattered, and the Zn and O elements are concentrated, and the element weight proportions of C, O, Zn and Pt in the figure are 57.98%, 29.94%, 9.39% and 2.69%, respectively, confirming that the sheet-shaped material synthesized is a zinc oxide nanosheet.

[0042] In conclusion, the present application shows that the Paenibacillus campinasensis (P. Paenibacillus kribbensis ) space mutant strain △PS04-17 has the ability to transform zinc nitrate hexahydrate and has a certain tolerance to zinc nitrate hexahydrate, and the zinc nitrate hexahydrate can be transformed into zinc oxide nanosheet by the strain △PS04-17, and the conversion rate is high; at the same time, the zinc oxide nanosheet is prepared by the biological synthesis of the strain △PS04-17, and the method is simple, safe, low in cost, friendly to the environment, and the prepared zinc oxide nanosheet has smooth surface, clear sheet structure, and orderly vertical or staggered folding, and can be applied in the fields of agriculture, industry, medical treatment and the like. The present application provides an efficient, green and rapid biological synthesis method for the biological synthesis of zinc oxide nanosheet.

[0043] The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. Paenibacillus chibensis (ATCC 13827) Paenibacillus kribbensis ) in the preparation of zinc oxide nanosheets, characterized in that, The strain has been preserved in Guangdong Microbial Culture Collection Center on March 17, 2022, and the preservation number is: GDMCC NO:62233.

2. A method of biosynthesizing zinc oxide nanoplatelets, characterized by, After activating the Paenibacillus kribbensis space mutant strain △PS04-17, it is inoculated into a fermentation medium containing zinc nitrate hexahydrate for fermentation culture, the fermentation liquid is centrifuged, the precipitate is collected, washed, centrifuged again, purified, freeze-dried to obtain; The strain △PS04-17 has been preserved in Guangdong Microbial Culture Collection Center on March 17, 2022, and the preservation number is: GDMCC NO:62233.

3. The method of claim 2, wherein, The inoculation amount of the strain is 2-5%.

4. The method of claim 3, wherein, The fermentation medium uses a modified Czapek culture medium, and the formula is: zinc nitrate hexahydrate 1-5 g / L, sucrose 25-30 g / L, potassium phosphate dibasic 1-1.3 g / L, magnesium sulfate heptahydrate 1-1.2 g / L, potassium chloride 0.5-0.8 g / L, ferrous sulfate heptahydrate 0.01-0.03 g / L, pH 6.5-7.

5.

5. The method of claim 2, wherein, The fermentation culture conditions are: temperature 25-28℃, rotation speed 100-200rpm or static culture, time 7-10 days.

6. The method of claim 2, wherein, The concentration of the seed liquid of the strain after activation is not less than 1 x 10 6 cuf / mL.

7. The method of claim 4 wherein, The concentration of zinc nitrate hexahydrate is 1.5-4.5 g / L.

8. The method of claim 2, wherein, The purification method is: first ultrasonic crushing, then extraction; the ultrasonic condition is set as power 400-500W, ultrasonic operation 3-5s, pause 3-5s, total time 30-40min.

9. Zinc oxide nanoplatelets characterized in that, Prepared by the method of any one of claims 2-8.

10. A product characterized by, Containing the zinc oxide nanosheet of claim 9.

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