http://www.paper.edu.cn

ARTICLES

Chinese Science Bulletin 2005 Vol. 50 No. 6 514—519 In this work, nano-MgO crystals with different parti- cle sizes prepared using different methods and reaction Influence of nano-MgO conditions were used to treat Bacillus subtilis var. niger. The effect of varying the particle size of the nano-MgO on particle size on bactericidal the bactericidal activity and the bactericidal mechanism were investigated. After being included in phenylpro- action against Bacillus subtilis pyl-type interior-wall paints in predetermined ratios, var. niger nano-MgO showed much better bactericidal activity against spores than nano-TiO2. , Dianqing, Yanjun, 1 Experimental David G. Evans & DUAN (i) Experimental materials. Mg(NO3)2·6H2O, Key Laboratory of Science and Technology of Controllable Chemical Na2CO3, Na2SO4, urea and ammonia were all AR grade Reactions, Ministry of Education, Beijing University of Chemical Tech- reagents. Phenylpropyl-type interior-wall paint was pur- nology, Beijing 100029, China chased from Beijing Lingzi Paint Chemical Company. Correspondence should be addressed to Duan Xue (e-mail: duanx@ mail.buct.edu.cn) Bacillus subtilis var. niger (spores, ATCC9372) was ob- tained from the Chinese Center for Disease Control and Abstract Nano-MgO with various particle sizes, synthe- Prevention in form of slice packages in accordance with 6 sized by different methods using Mg(NO3)2·6H2O, Na2CO3, international standards, and had a spore content of 10 Na2SO4, urea and ammonia solution as reactants, was used to cfu/piece (cfu=colony forming units). carry out bactericidal experiments on Bacillus subtilis var. (ii) Preparation of MgO with different particle sizes. niger. The results were compared with the effect of TiO , a 2 Known amounts of Mg(NO ) ·6H O and Na CO were common kind of photocatalytic material. The materials were 3 2 2 2 3 characterized by X-ray Diffraction (XRD), Transmission separately dissolved in distilled water. A magnesium car- bonate hydroxide precursor was prepared by mixing the Electron Microscopy (TEM), low temperature N2 adsorp- tion-desorption measurements and FT-IR, and the results two solutions by means of a newly-developed technique showed that the bactericidal ability of MgO increases with (method A) involving separate nucleation and aging decreasing particle size. Nano-MgO and an interior steps[5,6]. The precursor was then calcined at 550℃ for 1 wall-paint containing the material have better bactericidal h in order to produce the nano-MgO sample. effects than nono-TiO2 in both presence and absence of light. Using the same solutions as the above, magnesium The bactericidal mechanism is discussed. The surface of carbonate hydroxide precursors were prepared by drop − MgO can generate high concentrations of O2 which is wise addition of the salt solution to the alkali solution at highly active and can react with the peptide linkages in the controlled rates with vigorous stirring (method B). The coating walls of the spores. The spores are destroyed by the precursors were adequately washed and dried at 70℃ for resulting damage to their structure. 12 h, followed by calcination at 550, 750, 950℃ for 1 h Keywords: nano-particle, MgO, Bacillus subtilis var. niger, particle in order to produce the nano-MgO samples. size, sterilization. Mg(OH)2 and magnesium carbonate hydroxide pre- DOI: 10.1360/04wb0075 cursors were prepared by the ammonia method (method C) and the urea method (method D)[7] respectively. The pre- Nano-MgO has been widely used as a functional cipitates were separated and washed, and then dried at 90 [1,2] [3] material in various areas . Recently, it was reported ℃ for 1 h and calcined at 450℃ for 1 h in order to pro- that MgO had good bactericidal performance due to the duce the nano-MgO samples. − [8] formation of O2 anions at its surface in aqueous solution. A hydrothermal method (method E) with Mg [4] It was shown by the research work of Stoimenov et al. (NO3)2·6H2O, Na2SO4 and urea as reactants was also used that nano-MgO exhibits high bactericidal activity against to prepare magnesium carbonate, which was precipitated, bacteria, spores and viruses after adsorbing halogen gas washed, dried and calcined at 600℃ for 2 h in order to because of its large surface area, abundance of crystal de- produce nano-MgO. fects and positively-charged particles which can result in a (iii) Bactericidal tests. 0.50 g MgO bactericidal strong interaction with negatively-charged bacteria and tests prepared by different methods had contact with Ba- spores. cillus subtilis var. niger at 37℃ for 24 h. The number of Compared with TiO2, supported silver, supported surviving spores was counted after a follow-up cultivation copper and other kinds of solid bactericides, nano-MgO, of 48 h, and bactericidal efficiency was then calculated which is non-toxic and prepared from readily available according to the Chinese National Standard GB15981- materials, has great potential as a novel solid bactericidal 1995[9]. material under simple conditions. Therefore, further re- (iv) Interior-wall paint bactericidal test. Modified search into its bactericidal action is warranted. phenylpropyl-type interior-wall paints containing 5%

514 Chinese Science Bulletin Vol. 50 No. 6 March 2005

转载

中国科技论文在线 http://www.paper.edu.cn

ARTICLES

(weight percent) of nano-MgO or TiO2 were prepared and hydroxide precursor prepared by method B at 550, 750 quantitatively applied by brushing on 6 test plates. Test and 950℃ respectively increase with calcination tem- plates made contact with Bacillus subtilis var. niger at 37 perature, as shown by the increasing intensities and de- ℃ for 24 h in the presence and absence of light. The creasing FWHM values of the XRD peak. The average number of surviving spores was counted and the bacteri- particle sizes of the MgO samples estimated by the cidal efficiency was calculated according to the literature Scherrer Equation[11] are shown in Table 1. Because the method[10]. size of the magnesium carbonate hydroxide precursor par- (v) Analysis and characterization. Powder XRD ticles synthesized by the method A is small, the particle patterns were recorded on a Shimadzu XRD-6000 X-ray size of MgO-1 derived from this precursor is smaller than powder diffractometer (Cu Kα radiation, λ=0.15406 nm) that of MgO-2 obtained by method B. The particle sizes between 3° and 90°. The scan speed was 5°/min. The obtained from both TEM and the Scherrer Equation data crystal morphology was investigated using a Hitachi are the averages of primary particle sizes. The same values H-800 Transmission Electronic Microscope (TEM) in- obtained from the two methods show the same trend (Ta- strument. Low temperature N2 adsorption-desorption ex- ble 1). periments were carried out using a Quantachrome Auto- MgO-5 and MgO-6 samples were prepared by sorb-1 system. FT-IR characterization was performed on a method C with different concentrations of Mg(NO3)2. Bruker Vector 22 FT-IR spectrometer (as KBr discs, with Their XRD patterns exhibit intense, narrow peaks associ- the weight ratio of the sample to KBr of 1:100). ated with the formation of cubic crystals with large crystal 2 Results and discussion size. In has been suggested that the rate of reaction of ammonia solution with magnesium salts is fast so that (i) Controllable preparation of MgO with different [7] crystals of Mg(OH)2 grow rapidly . particle sizes. Eight MgO samples with different particle sizes were synthesized by methods A, B, C, D and E (see Table 1 Average particle size of MgO samples Preparation experimental section). As shown in Fig. 1, the patterns of Sample XRD/nm TEM/nm XRD peaks and their FWHMs (full width at half maxi- method mum) intensities vary with preparation methods. MgO-1 A 7.6 7.6 The XRD pattern of MgO-1, which was prepared by MgO-2 B 7.8 14.4 calcining a magnesium carbonate hydroxide precursor MgO-7 D 8.7 26.0 prepared by method A at 550℃ shows weak, broad MgO-3 B 22.4 35.9 diffraction peaks. As shown in the TEM micrograph of MgO-5 C 25.6 47.3 MgO-1 (Fig. 2), the average particle size of the crystallites MgO-4 B 28.1 56.3 is small. MgO-6 C 29.3 69.1 The particle sizes of samples MgO-2, MgO-3 and MgO-8 E 97.3 2145.9 MgO-4 prepared by calcining a magnesium carbonate

Fig. 1. XRD patterns of MgO nano-particles prepared by different methods.

Chinese Science Bulletin Vol. 50 No. 6 March 2005 515

中国科技论文在线 http://www.paper.edu.cn ARTICLES

Fig. 2. TEM micrographs of MgO samples synthesized by different methods. The rate of hydrolysis of urea can be controlled by calcining this precursor, the resulted MgO-8 sample gives adjusting the reaction temperature so that small particles narrow peaks in its XRD pattern, consistent with large of magnesium carbonate hydroxide with a narrow size particle size. distribution can be obtained. Sample MgO-7, obtained by (ii) Effect of particle size of MgO on bactericidal calcinations of the magnesium carbonate hydroxide pre- ability. Bacillus subtilis var. niger is a kind of spore with cursor, gives broad peaks in its XRD pattern consistent a dense coation and high resistance against chemicals. The with a small particle size. bactericidal activities of MgO with different particle sizes Magnesium carbonate with larger particle size can be against Bacillus subtilis var. niger were investigated in obtained under hydrothermal conditions (method E). After this work. The results are listed in Table 2, which shows

Table 2 Bactericidal efficiency of MgO with different particle sizes against B. subtilis var. niger Specific surface Ratio of number of Mg2+ ions on the surface Sample Size from TEM Bactericidal efficiency of B. niger (%) area/m2·g−1 to number in the bulk crystal (%) MgO-1 7.6 22.89 2.03 91.56 MgO-2 14.4 35.88 3.19 91.63 MgO-7 26.0 115.62 10.26 97.48 MgO-3 35.9 94.95 8.43 96.12 MgO-5 47.3 61.63 5.47 94.46 MgO-4 56.3 53.88 4.78 93.99 MgO-6 69.1 47.54 4.22 93.06 MgO-8 2145.9 20.43 1.81 75.71

516 Chinese Science Bulletin Vol. 50 No. 6 March 2005

中国科技论文在线 http://www.paper.edu.cn

ARTICLES that nano-MgO particles with different particle sizes ex- hibit excellent bactericidal activity against Bacillus sub- tilis var. niger since all the efficiencies are above 91%. In contrast, micron-sized MgO (MgO-8) shows only 76% bactericidal efficiency. The bactericidal efficiency against Bacillus subtilis var. niger decreased with increasing par- ticle size of nano-MgO for particles larger than 26 nm (as determined by TEM). The bactericidal efficiency against spores is 93% when the particle size of MgO is 69 nm, whereas when the particle size of MgO decreased to 26 nm, the efficiency reached 97%, but fell in to 91% with particles 8—14 nm in size. High bactericidal efficiency results from small crys- tallite size irrespective of the preparation methods as shown in Fig. 3. The efficiency rises smoothly as the par- ticles decrease from 69 to 47 nm in size, and then there is a more rapid increase, as the particle size decreases further. Fig. 4. Relationship between the size and surface bulk Mg2+ ratio. The specific surface areas determined from low tempera- ture N2 adsorption-desorption measurements on nano- (iii) Bactericidal activity of paint modified with MgO with different particle sizes are listed in Table 2. The nano-MgO. Adding nano-MgO with an average particle specific surface area decreased with increasing particle size of 26 nm to a phenylpropyl-type interior-wall paint size of the nano-MgO when the size is over 26 nm is low does not affect the chemical stability or shelf life of the for very small particles (<15 nm) as a result of aggrega- paint. Bactericidal tests against Bacillus subtilis var. niger tion effects. MgO belongs to the cubic crystal system and were carried out and the results are given in Table 3. The 2+ 2− ionic radii of Mg and O are 0.065 and 0.140 nm, re- bactericidal activity of normal interior-wall paint is rather 2+ spectively. The ratios of the number of Mg ions on the low, only 88%. Modification of the paint by addition of surface to that in the bulk crystal can be determined from nano-MgO results in enhanced bactericidal efficiency low temperature N2 adsorption-desorption measurements against spores, over 96%. It is clear that the addition of and are shown in Table 2. Fig. 4 shows the relationship nano-MgO can markedly improve the bactericidal ability 2+ between particle size and the ratio of number of Mg ions of interior-wall paints. on the surface to that in the bulk crystal. The ratio rises (iv) Comparison of bactericidal activity of nano- rapidly with decreasing size and there is a point of inflex- MgO and nano-TiO2. In recent years, photo catalytic [12,13] ion when the size is less than 48 nm. Therefore, bacteri- materials such as TiO2 have been widely studied . The [12] cidal efficiency increases with the increase in number of photo catalytic mechanism of TiO2 involves light- in- 2+ Mg ions on the surface and the decrease in particle size duced emission of excited electrons and the creation of of MgO. many positive holes which can react with surface oxygen and water to yield active oxygen and OH free radicals. Due to the oxidizing effect of these species, organic pol- lutants can be decomposed into carbon dioxide and water. A TiO2 sample with an average size of 29 nm was in contact with Bacillus subtilis var. niger under light irra- diation. The results are listed in Table 4, and clearly show that nano-TiO2 has a weak bactericidal activity against Bacillus subtilis var. niger, being only 68%. Compared with nano-TiO2, nano-MgO with an average particle size of 26 nm exhibits a better bactericidal capacity against spores, with 94% bactericidal efficiency.

Table 3 Comparison of bactericidal efficiency of unmodified and modified phenylpropyl-type interior-wall paints Bactericidal efficiency against Bacillus Paint subtilis var. niger (%) Untreated 88.86 Fig. 3. The effect of MgO particle size on MgO B. subtilis var. niger bactericidal efficiency. With 5% MgO (weight percent) 96.96

Chinese Science Bulletin Vol. 50 No. 6 March 2005 517

中国科技论文在线 http://www.paper.edu.cn ARTICLES

Table 4 Comparison of bactericidal efficiency of TiO2, MgO, and istic of the stretching vibration of C=O in amides. The modified phenylpropyl-type interior-wall paints N-H deformation peak occurs at 1544 cm−1, and the weak Bactericidal efficiency B. Sample Light irradiation −1 subtilis var. niger (%) peaks at 1242 and 1062 cm are associated with the

TiO2 yes 67.96 stretching vibrations of C-N and C-O, respectively. As MgO yes 94.46 shown in Fig. 5 curve 3, in the FT-IR spectrum of MgO Paint modified by mixed with spores after bactericidal experiments, the C=O yes 96.96 MgO stretching vibration is still present, but the intensity of the

Paint modified by TiO2 yes 96.25 C-O stretching vibration has increased dramatically. This Paint modified by no 96.15 indicates that chemical changes in coating of the spore MgO proteins have taken place. Paint modified by TiO2 no 93.11 It has been shown by Sawai et al. that MgO is very

readily hydrated and forms a layer of Mg(OH)2 on the Bactericidal experiments against Bacillus subtilis var. surface. Oxygen dissolved in the solution can produce niger were carried out with phenylproply-type inte- − [14] superoxide anions O2 by a single-electron reduction rior-wall paint containing nano-TiO2 with an average par- − ticle size of 29 nm and the results are also listed in Table 4. reaction and the O2 anions are stable in a basic envi- Paint modified by nano-TiO2 has almost the same bacteri- ronment. Since the surface of MgO has been covered with cidal effect under light irradiation as that modified by a layer of OH−, O− can exist on the surface at high con- nano-MgO. Their bactericidal efficiencies against Bacillus 2 subtilis var. niger are both above 96%. It was reported[12] centrations. The oxygen atom of C=O in secondary am- that photo catalytic by TiO2 is caused by light excitation ides has strong electronegativity, and the C atom is elec- − and weak UV light is needed. This reaction will not occur tron deficiency. It can be tentatively concluded that O2 in the absence of light irradiation, so that paint modified will attack the Cδ + atom and bond with it thus changing by TiO2 shows weaker bactericidal activity, only 93% the peptide linkage in the proteins. As a result of these without UV light irradiation. The paint modified by structural changes in the protein molecules the spores can nano-MgO has almost the same bactericidal activity be effectively destroyed by nano-MgO particles. against spores, above 96% under UV irradiation and in its Quantitative analysis of FT-IR was carried out on absence, which indicates that nano-MgO particles can mixtures of Bacillus subtilis var. niger and MgO with an greatly improve bactericidal performance of paint, even average size of 26 nm after different contact times. As without any UV light source. shown in Fig. 6, the peak area of the C-O stretching vibra- (v) Exploration of Bactericidal mechanism of MgO. tion increases with increasing contact time. This suggests Proteins in the coating of spores contain many peptide − O that O2 damages the secondary amide structure of the ‖ proteins of the spores, and the spores are gradually de- linkages such as secondary amide links (−C−NH−). The stroyed as a result. FT-IR spectrum of Bacillus subtilis var. niger is shown in Fig. 5 curve 1. The strong peak at 1638 cm−1 is character-

Fig. 6. FT-IR spectra of mixtures of MgO and B. subtilis var. niger Fig. 5. FT-IR spectra of B. subtilis var. niger (1), MgO (2), mixture of after different contact times. MgO and B. subtilis var. niger (3).

518 Chinese Science Bulletin Vol. 50 No. 6 March 2005

中国科技论文在线 http://www.paper.edu.cn

ARTICLES

From the bactericidal mechanism described above, it References − is clear that O2 with its strong oxidizability is the essen- 1. , S. Y., Magnesia sinter of high purity and density, Inorganic tial factor in the destruction of the spores. Small MgO Chemical Industry (in Chinese), 1994, 4: 19. particles have a large specific surface area and thus high 2. Zhang, L. D., , J. M., Nano-Meter Materials (in Chinese), OH－concentrations on their surface, and the increased Shenyang: Science Press, 1994, 303—306. O− concentration in the solution therefore results in a 2 3. Sawai, J., Kojima, H., Igarashi, H. et al., Antibacterial characteris- stronger interaction with the coating of the spores. The tics of magnesium oxide powder, World Journal of Microbiology & bactericidal activity of MgO is enhanced as a result and tends to increase with decreasing size of MgO. When the Biotechnology, 2000, 16: 187—194. particle size of MgO is rather small, aggregation effects 4. Stoimenov, P. K., Klinger, R. L., Marchin, G. L. et al., Metal oxide become very serious due to the very high surface energy nanoparticles as bactericidal agents, Langmuir, 2002, 18: 6679— of the particles. The increase in secondary size of MgO 6686. particles makes it difficult to make efficiency fully contact 5. , Y., Li, F., Evans, D. G. et al., Preparation of layered dou- with the spores so that bactericidal efficiency decreases. ble-hydroxide nanomaterials with a uniform crystallite size using a The formation of O− is not related to light irradiation, so 2 new method involving separate nucleation and aging steps, Chem- nano-MgO shows high bactericidal activity against spores istry of Materials, 2002, 14: 4286—4291. even in the absence of light. 6. Duan, X., Jiao, Q. Z., Li, L., China Patent, CN99119385.7, 1999. 3 Conclusions 7. , G. Z., , S. F., , G. L. et al., Synthesis of MgO (i) Nano-MgO with varying particle size distribu- nano-powder, Chinese Journal of Synthetic Chemistry (in Chinese), tions can be prepared by the method of separate nucleation 1996, 4: 300—302. and aging, the method of adding one solution dropwise into another at controlled rate speed with rapid agitation, 8. , Z. M., Li, F. Q., , P. H., Study on preparation of microcrys- the urea method and the ammonia method. tal magnesite by hydrothermal synthesis, Chinese Journal of Inor- (ii) The bactericidal efficiency decreased with de- ganic Chemistry (in Chinese), 2003, 19(8): 896—898. creasing size of MgO particles. When the size of MgO 9. Chinese National Standard, GB15981-1995. particles decreases from 69 to 26 nm, the number of Mg2+ 10. Li, Y. F., Wang, B. H., Hang, W. X. et al., Study on bactericidal ions on the surface increases, and the bactericidal effi- ciency increases from 93% to 97%. performance of nano-scale ZnO modified interior wall paint, Paint (iii) Interior-wall paints modified with nano-MgO and Coatings Industry (in Chinese), 2003, 33(8): 3—6. exhibit good bactericidal activity against spores, and the 11. , Y. L., , W. G., , D. J., Studies on preparation and anion bactericidal efficiency reaches 97%. exchange capacity of Mg-Fe-LDHs nanoparticles, Chemical Jour- (iv) Compared with nano-TiO , a common kind of 2 nal of Chinese Universities (in Chinese), 1999, 20: 1012—1016. photo catalytic material, nano-MgO has high bactericidal efficiency in both the presence and absence of light irra- 12. Zhao, D., Wang, J., Sun, B. H. et al., Development and application diation. of TiO2 photo- catalysts as antimicrobial agents, Journal of Liaon- (v) A mechanism is proposed on the basis of the re- ing University (Natural Sciences Edition) (in Chinese), 2000, 27: sults obtained. The surface of MgO can produce high 173—176. − concentrations of O2 which is highly active and can re- 13. , X. Y., Cheng, J. J., , Y. J., TiO2 photocatalytic antibacterial act with the peptide linkages in the coatings of the spores. materials, Glass & Enamel (in Chinese), 2000, 28(4): 42—47. Therefore the spores are destroyed by the resulting dam- 14. Baird, M. J., Lunsford, J. H., Catalytic sites for the isomerization of age to their structure. MgO with small particle sizes has 1-butene over magnesium oxide, Journal of Catalysis, 1972, 26: the strongest bactericidal activity. 440—450. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 90306012). (Received April 20, 2004; accepted July 8, 2004)

Chinese Science Bulletin Vol. 50 No. 6 March 2005 519