Biological composite material loaded with magnetic nanoparticles with core-shell structure, the preparation therefore and the application

Abstract

A preparation method of Bacillus subtilis biological composite material loaded with Fe.sub.3O.sub.4 magnetic nanoparticles with core-shell structure includes the following steps: 1) preparation of Fe.sub.3O.sub.4 nanoparticles, 2) preparation of Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles, 3) preparation of Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles; and 4) preparation of Bacillus subtilis@Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composite.

Claims

1. A preparation method of a Bacillus subtilis biological composite material loaded with Fe.sub.3O.sub.4 magnetic nanoparticles with core-shell structure, which comprises the following steps: 1) preparation of Fe.sub.3O.sub.4 nanoparticles: add ferrous sulfate heptahydrate and anhydrous sodium acetate to ethylene glycol according to the mole ratio of ferrous sulfate heptahydrate:anhydrous sodium acetate=1:5 to 8, stir until the ethylene glycol solution is transparent and transfer the ethylene glycol solution to a high pressure reaction vessel and the ethylene glycol solution is sealed for 5 to 8 hours at 150 to 200 DEG C., after centrifugation, washing and drying, obtain the Fe.sub.3O.sub.4 nanoparticles; 2) preparation of Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles: add the Fe.sub.3O.sub.4 nanoparticles obtained in step 1) to a mixture of ethanol and water, and disperse ultrasonically, under stirring, in accordance with the Fe.sub.3O.sub.4 nanoparticles:cetyltrimethylammonium bromide:tetraethoxysilane=1:3 to 5:2 to 3, add hexadecyltrimethylammonium bromide and tetraethoxysilane and keep at room temperature for 6 to 10 hours, after magnetic separation, washing and drying, obtain the Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles; 3) preparation of Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles: add the Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles obtained in step 2) to N,N-dimethylformamide, disperse by ultrasonic, and keep stirring, under the conditions of Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles:-aminopropyltriethoxysilane=50-100 mg:1 mL, add -aminopropyltriethoxysilane, stir overnight at room temperature, then centrifuge, wash and dry to obtain amino-modified Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles; add the above-mentioned amino-modified Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles into cyclohexanone and disperse by ultrasonic, under the conditions of inert gas protection and stirring, in accordance with the amino-modified Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles:4,4-azobis (4-4,4-azobis (4-cyanovaleryl chloride)=1:20-30 in mass ratio, add 4,4-azobis (4-4,4-azobis (4-cyanovaleryl chloride) to the above system, stir overnight at room temperature, and subject to magnetic centrifugation, washing and drying to obtain ABCPA modified Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles; add the ABCPA-modified Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles to cyclohexanone and disperse by ultrasonic, under the conditions of inert gas protection and stirring, in accordance with the nano-particles of Fe.sub.3O.sub.4@mSiO.sub.2 modified by ABCPA:N-acryloyloxysuccinimide=1:20 to 30 in mass ratio, and N-acryloyloxysuccinimide:4-vinylpyridine=1 g:10 to 15 mL, add N-acryloyloxysuccinimide and 4-vinylpyridine to the above system, reacting at 70-80 DEG C. for 0.5-1 hour, magnetic nanoparticles are obtained by magnetic centrifugation, washing and drying to obtain the Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nano-particles; 4) preparation of Bacillus subtilis@Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composite: add Bacillus subtilis to the PBS solution and dispers evenly, in accordance with the wet weight of Bacillus subtilis:Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nano-particles=50-100:1 in mass ratio, add the Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nano-particles obtained in step 3) to the above system, and placed on a constant temperature shaker at 30 C./120 rpm overnight, magnetic centrifuge, wash and dry to obtain the Bacillus subtilis biological composite material loaded with Fe.sub.3O.sub.4 magnetic nanoparticles with core-shell structure.

2. The preparation method according to claim 1, characterized in that: the molar ratio between the ferrous sulfate heptahydrate and anhydrous sodium acetate described in step 1) is 1:7.

3. The preparation method according to claim 1, which characterized in: the volume ratio of ethanol to water in the mixture of ethanol and water in step 2) is 4:1; the mass ratio of the Fe.sub.3O.sub.4 nanoparticles, cetyltrimethylammonium bromide, tetraethoxysilane described in the step 2) is 1:3:2.3.

4. The preparation method according to claim 1, which characterized in: the ratio between the Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles and the aminopropyltriethoxysilane described in the step 3) is 200 mg:3 mL; the mass ratio of the amino-modified Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles to 4,4-azobis (4-cyanovaleryl chloride) in step 3) is 1:20; the weight ratio between the ABCPA-modified Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles and the N-acryloyloxysuccinimide described in step 3) is 1:20; the ratio between N-acryloyloxysuccinimide and 4-vinylpyridine in step 3) is 1 g:10 mL.

5. The preparation method of claim 1, which characterized in: the inert gas in step 3) is selected from any of nitrogen, helium, and argon.

6. The preparation method according to claim 5, which characterized in: the inert gas in step 3) is nitrogen.

7. The preparation method according to claim 1, which characterized in: the mass ratio between the wet weight of Bacillus subtilis described in step 4) and the Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles is 100:1.

8. The preparation method according to claim 1, which characterized in: in step 4), the pH of said PBS solution is 7.

9. A Bacillus subtilis biological composite material loaded with a Fe.sub.3O.sub.4 magnetic nanoparticle having a core-shell structure prepared by the preparation method according to claim 1.

10. A method of treating waste water containing hexavalent chromium comprising applying the Bacillus subtilis biological composite material loaded with a Fe.sub.3O.sub.4 magnetic nanoparticle having a core-shell structure of claim 9 to the waste water.

Description

APPENDED DRAWINGS

[0029] FIG. 1 shows the transmission electron microscopy (TEM) of Fe.sub.3O.sub.4 nanoparticles.

[0030] FIG. 2 shows the transmission electron microscopy (TEM) of Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles.

[0031] FIG. 3 shows the transmission electron microscopy (TEM) of Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles.

[0032] FIG. 4 is a scanning electron microscopy (SEM) image of Bacillus subtilis before and after treatment of Cr (VI).

[0033] FIG. 5 shows the transmission electron microscopy (TEM) of the B. subtilis@Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composite.

[0034] FIG. 6 is the degradation curve of Cr (VI) and the OD600 curve of B. subtilis.

[0035] FIG. 7 shows the UV-Vis absorption spectra of Cr (VI) treated with B. subtilis at different times.

[0036] FIG. 8 shows the degradation of Cr (VI) by Bacillus subtilis, Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles and Bacillus subtilis @Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composite.

DETAILED DESCRIPTION

[0037] The following will be combined with the appended drawings and the implementation of the specific case to make further explanation of the invention.Unless specifically noted that agents used in the following example, experimental material and equipment, etc. Can be obtained through commercial means.

EXAMPLE 1

Preparation of Fe.SUB.3.O.SUB.4 .Nanoparticles

[0038] In this experiment, all the chemical agents were of analytical grade and were used without further purification. The spherical magnetic particles were prepared according to the literature with some modification..sup.40 As usually, 2.02 g of Fe(NO.sub.3).sub.3.9H.sub.2O and 4.1 g of sodium acetate were dissolved in 50 mL of ethylene glycol (EG) with stirring for 30 min. The obtained solution was transferred to a Teflon-lined stainless-steel autoclave and heated at 180 C. for 6 h. Then the autoclave was naturally cooled to room temperature. The gained black magnetite particles were washed with ethanol for several times, and dried in vacuum at 60 C. for 5 h.

[0039] FIG. 1 shows the TEM of Fe.sub.3O.sub.4 nanoparticles. It can be seen, Fe.sub.3O.sub.4 nanoparticles dispersed and diameter of about 30 nm.

EXAMPLE 2

Preparation of Fe.SUB.3.O.SUB.4.@mSiO.SUB.2 .Nanoparticles

[0040] The core-shell structured Fe.sub.3O.sub.4@mSiO.sub.2 microspheres were prepared through a modified Stber method. In a typical process, 0.10 g of obtained Fe.sub.3O.sub.4 particles were treated using 0.1 M HCl solution by ultrasonication for 20 min. Whereafter, the treated Fe.sub.3O.sub.4 particles were separated via centrifugation, washed with deionized water. At the same time, The Fe.sub.3O.sub.4 was dispersed in the mixture solution of 80 mL of ethanol, 20 mL of deionized water, and 1.0 mL of concentrated ammonia aqueous solution (28 wt. %). Afterward, 0.3 g of cetyltrimethylammonium bromide (CTAB) was added dropwise to the solution. After this, 0.25 mL TEOS was added dropwise into the solution under vigorous stirring for 6 h. After reaction for 6 h, the product was collected by magnetic separation and tautologically washed with ethanol and deionized water. The above coating process was redone twice. The structure-directing agent (CTAB) was removed with ethanol and deionized water for three times. The obtained precipitate was separated and washed with deionized water. Subsequently, the product was dried in vacuum at 60 C. for 24 h. The manufactured microspheres what was called Fe.sub.3O.sub.4@mSiO.sub.2.

[0041] FIG. 2 shows the TEM of Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles. It can be seen that Fe.sub.3O.sub.4 surface coated with mesoporous silica (mesoporous silicon dioxide, abbreviated as mSiO.sub.2), and has good dispersion, the diameter of nanoparticles increased to 50 nm.

EXAMPLE 3

Preparation of Fe.SUB.3.O.SUB.4.@mSiO.SUB.2.@MANHE Nanoparticles

[0042] 200 mg (Fe.sub.3O.sub.4@mSiO.sub.2) of the nanoparticles obtained for 250 ml flask, the flask to add 150 ml acetonitrile, ultrasound 30 min, then add 3 ml KH550, mechanical agitation for the night.

[0043] The acid chloride derivative of ABCPA (ClABCPA) was prepared by a reaction of ABCPA and PCl.sub.5. ABCPA (3.0 g) was dissolved in dichloromethane (25 mL) and cooled to 0 C. PCl.sub.5 (24 g) in 25 mL of CH.sub.2Cl.sub.2 was added into the above solution and stirred overnight. After the reaction, the excess PCl.sub.5 was removed by filtration. The clear solution was added into 5-fold of hexane at 0 C., and 4,4-azo-bis(4-cyanopentanoicchloride) was obtained after filtration. Fe.sub.3O.sub.4mSiO.sub.2NH.sub.2 nanoparticles (0.600 g) were added to 80 mL of dry dimethylformamide. After 0.5 h of ultrasonication, Fe.sub.3O.sub.4@mSiO.sub.2NH.sub.2 (0.60 g) was dispersed in a mixture of 80 mL of CH.sub.2Cl.sub.2 and 2 mL of triethylamine, and ClABCPA (2.5 g) in 25 mL of dry CH.sub.2Cl.sub.2 was added to the dispersion. After stirring at 0 C. for 2 h, the dispersion was stirred at room temperature overnight. Fe.sub.3O.sub.4@mSiO.sub.2ABCPA was obtained after filtration and washing with methanol and dichloromethane. Polymer on Fe.sub.3O.sub.4@mSiO.sub.2ABCPA sheets were prepared by free-radical polymerization. In a Schlenk flask, Fe.sub.3O.sub.4@mSiO.sub.2ABCPA (0.05 g), 4-vinylpyridine (4 mL) and N-Acryloxysuccinimide (0.7 g) monomer were dissolved in 9 mL of Cyclohexanone. After 0.5 h min sonication, the dispersion was stirred at 75 C. for 5 h. The resulting product was dissolved in acetone and centrifuged to remove the free polymer chains which were not anchored to the nanoparticles. The final product (FSM) was dried in vacuum at 50 C.

[0044] FIG. 3 shows the TEM of Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles. As can be seen from the figure, Fe.sub.3O.sub.4@mSiO.sub.2 nanoparticles coated with a layer of polymer (MANHE), dispersion decreased, the diameter of nanoparticles to further increase to 100 nm.

EXAMPLE 4

Culture of Bacillus subtilis and Degradation of Cr (VI)

[0045] B. subtilis ATCC-6633 was obtained from Fujian Institute of Microbiology, China. Previous study suggested that the maximum hexavalent chromium resistance of B. subtilis ATCC-6633 is 40 mg/L. Planktonic cells were grown at 30 C. with shaking (120 rpm) for 48 h in modified LuriaeBertani (LB) liquid medium (pH=7) supplemented with 5 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract and 5 g/L glucose.

[0046] FIG. 4a shows the SEM of Bacillus subtilis Cr (VI), FIG. 4b shows the SEM of Bacillus subtilis after Cr (VI) degradation. From the figure, it can be seen that the surface of the cell before Cr (VI) was smooth and the surface of the cell after Cr (VI) was irregular.

EXAMPLE 5

Preparation of Bacillus subtilis@Fe.SUB.3.O.SUB.4.@mSiO.SUB.2.@MANHE Composite

[0047] After strain B. subtilis ATCC-6633 was cultured for 48 h in 100 mL LB medium with shaking (120 rpm), the B. subtilis ATCC-6633 were harvested by centrifugation (5 min, 5500 g) and washed twice with PBS (sterile phosphate buffer solution). Then, the cell pellets were resuspended in PBS. Subsequently, 30 mg FSM was added into above system. Planktonic cells were cultivated at 30 C. with shaking (120 rpm) for 5 h. After that, BFSM were obtained by magnetic separation.

[0048] FIG. 5 shows the TEM of a B. subtilis@Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composite. It is clear from the figure that Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles are modified to the surface of the cell.

EXAMPLE 6

Degradation of Cr (VI) (Concentration 40 ppm) by Bacillus subtilis and Test Results

[0049] The bacterial cells were collected by centrifugation and re-dispersed in waste water (100 mL) containing 40 ppm Cr (VI) and sampled at different times. The concentration of the solution at each time was measured by colorimetry and the UV- Spectrum.

[0050] FIG. 6 shows the degradation curve and OD600 curve of Cr (VI) treated by Bacillus subtilis. It can be seen that the concentration of Cr (VI) decreased and the OD600 of bacteria increased on the original basis, which indicated that Bacillus subtilis could tolerate Cr (VI) VI) can effectively survive in Cr (VI)containing water. FIG. 7 shows the UV-Vis absorption spectra of Cr (VI) treated with Bacillus subtilis at different times. It can be seen that at 364 nm, the maximum absorption wavelength decreases with time, which indicates that the concentration of Cr (VI), And no absorption peak after 120 h. At 364 nm, the concentration of Cr (VI) in the surface solution has almost reached zero.

EXAMPLE 7

Effect Comparison of Treatment of Cr (VI) (Concentration 40 ppm) with Bacillus subtilis, Fe.SUB.3.O.SUB.4.@mSiO.SUB.2.@MANHE Nanoparticles, Bacillus subtilis@Fe.SUB.3.O.SUB.4.@mSiO.SUB.2.@MANHE Composite

[0051] Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles and Bacillus subtilis@Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composites were weighed and dispersed in 40 ppm Cr (VI) solution. Samples were taken at different times and samples were taken and the corresponding UV-Vis absorption spectra were plotted.

[0052] FIG. 8 shows the degradation of Cr (VI) by Bacillus subtilis, Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticles and Bacillus subtilis@Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composite. It is clear from the comparison that Bacillus subtilis@Fe.sub.3O.sub.4@mSiO.sub.2@MANHE composite has the highest removal rate of hexavalent chromium and the best effect.

[0053] In summary, the present invention realizes the degradation of Cr (VI) by adsorption on the side of Fe.sub.3O.sub.4@mSiO.sub.2@MANHE nanoparticle to Bacillus subtilis, and has high degradation speed and high removal efficiency. More importantly, to achieve the magnetic separation of bacteria to solve the obstruction of microbial treatment of heavy metal pollution application problems. In addition, the production method disclosed in the present invention is easy to handle and the raw materials used are inexpensively available. Therefore, the magnetic nanometer biological composite material of the invention will have good application prospect in the future sewage treatment.