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Antibacterial activated carbons prepared from pitch containing organometallics
Carbon 39 (2001) 1963–1969
Antibacterial activated carbons prepared from pitch containing organometallics Hisashi Tamai a , Nobuyoshi Katsu a , Kazuhisa Ono b , Hajime Yasuda a , * a
b
Department of Applied Chemistry, Faculty of Engineering, Hiroshima University, Higashi-hiroshima 739 -8527, Japan Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1 -4 -1 Higashi-hiroshima 739 -8527, Japan Received 19 July 2000; accepted 29 December 2000
Abstract A simple method for the preparation of antibacterial activated carbons was reported. The antibacterial activated carbons uniformly contain metal oxides, i.e. CaO, MgO, NiO, CoO and ZnO. These activated carbons were readily prepared by steam activation of pitch containing organometallics, i.e. CaI 2 , t-BuMgBr, Ni(acac) 2 , Co(acac) 3 , and Zn(acac) 2 . The antibacterial activities for B. subtilis, S. aureus, and E. coli were estimated by the growing inhibitory effect in the solutions containing activated carbons and by haloes at surrounding of activated carbons on agar. The CaO, MgO, CoO, and ZnO dispersed activated carbons exhibited obviously the antibacterial activity for B. subtilis both in the solutions and on agar. The MgO and CoO dispersed ones exhibited a growing inhibition effect for S. aureus. On the other hand, no inhibitory effect was observed for the growing of E. coli. 2001 Elsevier Science Ltd. All rights reserved. Keywords: A. Activated carbon, pitch; B. Activation
1. Introduction Activated carbons have attracted much attention as one of the effective adsorbents. For wide applications of activated carbons as adsorbents, the controlling of pore size and its distribution is important. Until a recently there have been many investigations on the controlling of pore size. Recently, we have developed highly mesoporous carbons by activating pitch containing rare earth metal complexes with steam [1–3], and then we showed that metal or metal oxide particles were uniformly dispersed in activated carbons obtained from pitch containing organometallics in terms of electron probe microanalysis (EPMA) and X-ray diffraction (XRD) analyses [3,4]. On the other hand, the antibacterial activity of activated carbons are also very important for application in water purification systems, e.g. purification of drinking water because the growing of bacteria on the surface of activated carbons spoils the function of activated carbon and resulting water may be polluted by bacteria. In general, the antibacterial activities of activated carbons are available as *Corresponding author. Tel.: 181-824-24-7731; fax: 181824-22-7191. E-mail address: [email protected] (H. Yasuda).
Ag deposited activated carbons prepared by impregnation methods [5–7]. However, in the case of Ag supported activated carbons prepared by impregnation or deposition methods, there arise some problems, e.g. needing a few steps for Ag-supporting such as impregnation in Ag solutions and heat-treatment of activated carbons, a decrease in surface area and pore size by deposited Ag, and rapid elution of Ag ions at the initial stage of usage [5]. Sawai et al. [8–10] reported that some metal oxides powders e.g., MgO, CaO, and ZnO showed a growth inhibitory effect for bacteria such as Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis). Based on these results., we expected that metal oxides particles uniformly dispersed in activated carbons should show antibacterial activity. Especially, finely and uniformly dispersing of metal oxide particles in activated carbons plays an important role to realize the excellent antibacterial effect. From these points of view, we attempted the preparation of the antibacterial activated carbons without complicated steps used for the preparation of Ag-supporting activated carbon. We supposed the formation of metal oxides simultaneously with the formation of pores in activated carbons by activating pitch containing organometallics. The obtained activated carbons exhibited antibacterial
0008-6223 / 01 / $ – see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S0008-6223( 01 )00003-3
1964
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
activities for B. subtilis and S. aureus, although their activities were lower than that of Ag deposited activated carbons. We show that the antibacterial activated carbons can be prepared by the simple procedure.
2. Experimental
2.1. Materials Pitch was coal tar pitch from Osaka gas Co. (softening point: 72.28C, C: 93.2%, H: 4.7%, N: 1.1%). CaI 2 , tBuMgBr, Ni(acac) 3 , Fe(acac) 3 , Co(acac) 3 , Al(acac) 3 , and Zn(acac) 2 were purchased from Kanto Chemical and used without further purification. Manganocene (Cp 2 Mn) was synthesized as follows [11]: cyclopentadiene (1.59 mmol) was added slowly to sodium shot (3.65 g) in THF (100 ml), and then to this solution manganous chloride (10 g) was added. The mixture was refluxed for 3 h and THF was removed under vacuum. The resulting Cp 2 Mn was sublimed in vacuo. Dark brown Cp 2 Mn crystals were obtained (yield; 2 g). Tetrahydrofuran (THF) (Kanto Chemical) was dried over Na / K alloy and distilled before use. S. aureus (HUT005), B. subtilis (IFO3134), and Escheritia coli (E. coli) (HUT8106) were obtained from Fermentation Technology Department of Hiroshima University.
2.2. Carbons Various activated carbons were prepared by steam activation of pitch containing organometallics. Pitches containing organometallics were obtained by mixing a THF solution of pitch with THF solutions of organometallics. A typical preparation method is as follows: 4.79 g of pitch was placed in a 300 ml two necked round bottom flask and volatile components were removed under reduced pressure at 1208C. The pitch was dissolved in 90 ml of dry THF. Zn(acac) 2 (1.209 g) dissolved in 90 ml of dry THF was added to the pitch solution. After stirring for 2 h, THF was removed by flash distillation. Activated carbons were obtained by activation of pitch containing organometallics with steam at 9008C. Ag supported activated carbons were prepared by the impregnation method [6]. Activated carbon prepared from organometallics-free pitch by steam activation was impregnated in aq. solutions of AgNO 3 for 24 h. After evaporation of AgNO 3 solution, the activated carbon was heat-treated at 4208C for 1 h.
2.3. Methods Specific surface area and pore size distribution were determined by BET method, and t-plots (for micropore) and BJH [12] (for mesopore) methods, respectively, by applying N 2 adsorption isotherms measured using a Quantachrome Autosorb-6. XRD analyses were performed using
a Rigaku RDA-1B system with Cu Ka radiation. The dispersion of metal oxides in activated carbons was observed by electron probe microanalysis (EPMA) with a JEOL JXA-8900. The metal contents in carbons were measured by particle-induced X-ray emission (PIXE) analysis and ash contents in activated carbons. PIXE analysis was performed using a van de Graaff accelerator of Hiroshima University. The intensity of the characteristic X-ray generated by irradiation of H 1 was measured. We estimated antibacterial activities of metal oxides particles dispersed in activated carbons by the growing inhibitory effect for B. subtilis, S. aureus, and E. coli. A growing inhibitory effect was determined by two kinds of methods; (1) measurement of bacteria growing in the solutions containing activated carbons as follows: a portion of B. subtilis was inoculated in 5 ml of sporulation medium (beef extract 10 g, poly-pepton 10 g, NaCl 5 g / 1 l of pure water). The medium was incubated at 378C for 18 h. The spores were inoculated in 5 ml of sporulation medium and to this medium activated carbons were added. The spores were filtered after the prescribed time. The filtrate (0.5 ml) was diluted by 4.5 ml of sporulation medium. The bacteria growing was determined by measuring optical density of the diluted solution. Optical density (OD 660 ( l 5660 nm)) was measured by UV–Visible spectrometer(Pharamacia LKB Biochrome 4060 UV–Visible Spectrometer). (2) Measurement by the halo method as follows [12]: bacteria was inoculated in 5 ml of a sporulation medium (poly-pepton 10 g, yeast extract 2 g, and magnesium sulfate 1 g / 1 l of pure water), and this solution was incubated at 378C for 18 h. The spores were inoculated in 100 ml of sporulation medium and incubated at 378C for 18 h. Agar containing these spores was placed onto agar plates containing activated carbons and dried for a few minutes. After incubating for 24 h at 378C, the lengths of haloes formed surrounding the activated carbons were measured.
3. Results and discussion Table 1 shows the carbonized yields and pore characteristics of the activated carbons obtained from pitches containing various organometallics. The addition of CaI 2 lowered the yield and BET specific surface area. The formed CaO accelerated the consumption of carbon precursors such as pitch during steam activation and as a result, the formed pores were supposed to be destroyed throughout activation. On the other hand, the addition of other organometallics, i.e. t-BuMgBr, Zn(acac) 2 , Ni(acac) 3 , Fe(acac) 3 , Co(acac) 3 , and Al(acac) 3 into pitch promotes the formation of pores and BET specific surface areas are higher than that from organometallics-free pitch in spite of the short activation time. Especially, the promoting effect for pore formation is remarkable in the addition of
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
1965
Table 1 Preparation of activated carbons from pitches containing various organometallics a Sample
Organometallics
Metal in pitch (wt.%)
Activation time (min)
Yield (%)
BET surface area (m 2 / g)
Mean pore size (nm)
Ca–AC Mg–AC Zn–AC Mn–AC Ni–AC Fe–AC Co–AC Al–AC AC
CaI 2 t-BuMgBr Zn(acac)2 Cp 2 Mn Ni(acac) 2 Fe(acac) 3 Co(acac) 3 Al(acac) 3 –
5.0 5.0 5.0 2.5 5.0 5.0 5.0 5.0 –
3 15 15 25 12 15 18 18 30
14.8 40.2 26.3 31.8 28.2 36.9 24.6 32.0 24.7
0.4 118 150 76 131 159 159 221 88
50.0 3.1 2.6 3.6 3.2 3.8 3.8 2.2 2.5
a
Activation temp.: 9008C.
Co(acac) 3 and Al(acac) 3 . Fig. 1 shows the pore distributions of activated carbons obtained from pitches containing t-BuMgBr and Zn(acac) 2 . Micropores of about 0.5 nm of pore radius were mainly developed in activated carbon obtained from pitch containing t-BuMgBr, whereas pores of large size (mesopores) were formed in addition to micropores by the addition of Zn(acac) 2 . Similarly, a large ratio of micropores and small ratio of mesopores were formed in activated carbons obtained from pitches containing other organometallics. The decomposition of organometallics results in the formation of metal or metal oxide in the course of activation of pitch [2,4]. We measured metal contents in the activated carbons obtained. The results are shown in Table 2. Metal contents in the activated carbons range from 4.3 to 9.8 wt.% in the cases of Zn, Ni, Fe, Co, and Ni. These values were lower compared with the theoretical values calculated from metal contents in pitch (5 wt.%). A part of metal atoms is supposed to disappear throughout the decomposition of organometallics during activation. On the other hand, the detection of light atoms such as Ca
and Mg by PIXE analysis was impossible. Based on metal contents determined from ash contents in activated carbons obtained from pitches containing CaI 2 and t-BuMgBr, it is supposed that most metal atoms remain in the activated carbons. In addition, XRD analysis indicated the formation of crystals of CaO and MgO in the activated carbons as described below. Sawai et al. [8–10] reported that some metal oxides showed antibacterial activities. We analyzed metal species formed from organometallics in activated carbons during activation by XRD analyses. Fig. 2 shows XRD patterns of the activated carbons obtained from pitches containing CaI 2, t-BuMgBr, Zn(acac) 2 , and Cp 2 Mn. The peaks due to CaO, MgO, and ZnO on each XRD patterns were observed in addition to the broad peak of carbon crystallites. As shown in Fig. 2, MnO and Mn 2 O 3 particles were formed from Cp 2 Mn. Similarly, the formation of metal oxides, i.e. Al 2 O 3 , and Fe 2 O 3 from Al(acac) 3 and Fe(acac) 3 , respectively, was observed. On the other hand, the formation of metal particles, i.e. Co and Ni in addition to metal oxides such as CoO and NiO from Co(acac) 3 and Ni(acac) 3 , was observed. Table 2 Metal content in activated carbons obtained from pitches containing organometallics
Fig. 1. Pore distributions of activated carbons obtained from pitches containing t-BuMgBr and Zn(acac) 2 (s); t-BuMgBr, (d); Zn(acac) 2 .
Sample
Metal in pitch (wt.%)
Metal in carbon (wt.%)
Ca–AC Mg–AC Zn–AC Mn–AC Ni–AC Fe–AC Co–AC Al–AC
5.0 5.0 5.0 2.5 5.0 5.0 5.0 5.0
25.0 a 9.9 a 4.4 b 0.8 b 9.8 b 6.5 b 8.6 b 4.3 b
a b
Determined from ash content. Determined by PIXE.
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H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
Fig. 2. XRD patterns of activated carbons obtained from pitches containing organometallics (a), CaI 2 ; (b), t-BuMgBr; (c), Zn(acac) 2 ; (d), Cp 2 Mn.
Fig. 4. Optical densities of sporulation medium containing Ca– AC for B. subtilis. Concentration of activated carbon (d); Ca–AC 10 mg / ml, (m); Ca–AC 30 mg / ml, (n); AC 30 mg / ml, (s); none.
The dispersion states of metal oxides in the activated carbons obtained from pitches containing organometallics are important for antibacterial activity of activated carbons. The homogeneous dispersion of metal oxides were observed by EPMA. SEM and EPMA images of the activated carbon obtained from pitch containing Zn(acac) 2 are exemplified in Fig. 3. ZnO were dispersed almost uniformly in activated carbon. The distributions of other metal oxides were also uniform as observed for ZnO. The antibacterial activities for B. subtilis, S. aureus, and
E. coli were investigated. Bacteria were incubated in sporulation medium containing activated carbons and the change of optical density (OD) of medium with incubation time was measured. Figs. 4–7 show the results on the activated carbons obtained from pitches containing CaI 2 , t-BuMgBr, Zn(acac) 2 , and Cp 2 Mn, for B. subtilis. As shown in Figs. 4 and 5, in the cases of the activated carbons obtained from pitches containing CaI 2 and tBuMgBr, no increase of OD with time was observed while
Fig. 3. EPMA image of activated carbon obtained from pitch containing Zn(acac) 2 .
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
Fig. 5. Optical densities of sporulation medium containing Ma– AC for B. subtilis. Concentration of activated carbon (d); Mg– AC 10 mg / ml, (m); Mg–AC 30 mg / ml, (j); Mg–AC 50 mg / ml, (n); AC 30 mg / ml, (s); none.
the OD of medium without activated carbon or the OD of medium containing activated carbon obtained from organometallics-free pitch increased with incubation time. These results obviously indicate that the activated carbons obtained from pitches containing CaI 2 and t-BuMgBr possess strong antibacterial activities for B. subtilis. In addition, the activated carbons obtained from pitches containing Co(acac) 3 and Ni(acac) 3 showed the same antibacterial effect for B. subtilis as that from pitch containing t-BuMgBr. As shown in Fig. 6, the OD of sporulation medium for activated carbon containing ZnO slightly increases at the initial stage of incubation and the value of OD decreased with increasing the amount of
Fig. 6. Optical densities of sporulation medium containing Zn– AC for B. subtilis. Concentration of activated carbon (m); Zn–AC 30 mg / ml, (d); Zn–AC 50 mg / ml, (n); AC 30 mg / ml, (s); none.
1967
Fig. 7. Optical densities of sporulation medium containing Mn– AC for B. subtilis. Concentration of activated carbon (d); Mn– AC 30 mg / ml, (m); Mn–AC 50 mg / ml, (n); AC 30 mg / ml, (s); none.
activated carbon added in incubation medium. The ZnO dispersed activated carbon is supposed to suppress growing of bacteria, although the antibacterial activity was lower than that of the MgO dispersed one. On the other hand, as shown in Fig. 7, the activated carbon containing MnO and Mn 2 O 3 exhibited no growing inhibitory effect for B. subtilis. Fig. 8 shows the changes of ODs of sporulation medium containing activated carbons for S. aureus with incubation time. Mediums containing activated carbons prepared from pitch containing Zn(acac) 2 or metal oxide-free activated carbon exhibited the same growing curves as that in medium without activated carbon. There is no antibacterial activity in activated carbon containing ZnO for S. aureus.
Fig. 8. Optical densities of sporulation medium containing 30 mg / ml of activated carbons for S. aureus (j); Ca–AC, (s); Mg–AC, (n); Zn–AC, (h); Ni–AC, (m); AC, (d); none.
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
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Fig. 9. Optical densities of sporulation medium containing 30 mg / ml of activated carbons for E. coli (j); Ca–AC, (s); Mg– AC, (n); Zn–AC, (h); Ni–AC, (앳); Co–AC, (m); AC, (d); none.
On the other hand, the increase in ODs of mediums containing the NiO, MgO, and CaO dispersed activated carbons is low, compared with the OD of medium containing metal oxide-free activated carbon. These activated carbons exhibited an inhibitory effect for the growing of S. aureus. On the antibacterial activity for E. coli, Fig. 9 shows the relation between ODs of sporulation mediums containing activated carbons and incubation time. ODs of mediums containing metal oxides dispersed activated carbons scarcely increased with time. However, the OD of medium containing metal oxide-free activated carbons was also low. The growing inhibitory effect of the metal oxides dispersed activated carbons for E. coli seems due to the adsorption of E. coli on activated carbons rather than the antibacterial activity of metal oxides. Furthermore, in order to clarify the antibacterial activity of metal oxides dispersed activated carbons, we measured the growing inhibitory effect on agar by the halo method. Table 3 shows the lengths of haloes formed surrounding the activated carbons. The activated carbons containing
Table 3 Growth inhibitory effect for bacteria on agar Sample
Ca–AC Mg–AC Zn–AC Ni–Ac Fe–AC Co–AC Al–AC AC–Ag5 AC–Ag10
CaO, MgO, ZnO, and CoO show the formation of haloes of almost the same lengths for B. subtilis. This formation of haloes indicates that these activated carbons give the inhibitory effect for the growth of B. subtilis. However, the lengths of haloes are half of that by Ag supported activated carbons, which were prepared by the impregnation method using aq. solution of AgNO 3 . On the other hand, NiO dispersed activated carbon for B. subtilis and S. aureus and CaO dispersed activated carbon for S. aureus, no growing inhibitory effect was observed on agar in spite of the fact that it exhibited the antibacterial activity in the solutions. This may be due to the difference in contact area of activated carbons to bacteria. The contact area of activated carbon in solutions is supposed to be larger than that on agar. These results suggest that the antibacterial activity of NiO dispersed activated carbon is not so strong as that of MgO dispersed activated carbon.The activated carbons obtained from pitches containing organometallics showed no inhibitory effect on agar for E. coli, in spite of that Ag supported activated carbon exhibited strong inhibitory effect.
4. Conclusion The antibacterial activated carbons can be simply prepared by steam activation of pitch containing organometallics. Metal oxides were formed uniformly in activated carbons and the antibacterial activities were dependent on the kind of metal oxides. The CaO, MgO, CoO, and ZnO dispersed activated carbons obtained from pitches containing CaI 2 , t-BuMgBr, Co(acac) 3 , Ni(acac) 2 , and Zn(acac) 3 , respectively, exhibited good antibacterial activities for B. subtilis. The MgO and CoO dispersed ones presented a growing inhibitory effect in sporulation solutions and on agar for S. aureus. On the other hand, no inhibitory effect was observed for the growing of E. coli. The antibacterial activated carbons do not need the complicated procedures such as that for the preparation of Agsupporting activated carbon.
Acknowledgements
Halo length (mm) B. subtilis
E. coli
S. aureus
4.2 4.0 4.2 0 0 4.5 0 9.9 9.9
0 0 0 0 0 0 0 4.0 6.0
0 8.5 0 0 0 6.5 0 9.0 13.3
This work is partially supported by a Grant-in-aid for ‘Research for the Future Program’, Nano-carbon, from the Japan Society for the Promotion of Science.
References [1] Tamai H, Kakii T, Hirota Y, Kumamoto T, Yasuda H. Chem Mater 1996;8:454–62. [2] Tamai H, Kojima S, Ikeuchi M, Mondori J, Kanata T, Yasuda H. Tanso 1996;1996:243–8.
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969 [3] Tamai H, Ikeuchi M, Kojima S, Yasuda H. Ad Mater 1997;9:55–8. [4] Tamai H, Kataoka Y, Nishiyama F, Yasuda H. Carbon 2000;38:899–906. [5] Oya A, Yoshida S. Carbon 1996;34:53–7. [6] Li CHY, Wan YZ, Wang J, Wang YL, Jiang XQ, Han LH. Carbon 1998;36:61–5. [7] Wan YZ, Wang YL, Wen TY. Carbon 1999;37:351–8. [8] Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem Eng Jpn 1995;28:288–93.
1969
[9] Sawai J, Saito I, Kanou F, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem Eng Jpn 1995;28:352–4. [10] Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. J Chem Eng Jpn 1995;28:556–61. [11] Wilkinson G, Cotton FA, Birmingham JM. J Inorg Nucl Chem 1956;2:95–113. [12] Barrett EP, Joyner LS, Halenda PP. J Am Chem Soc 1951;73:373–7.
Antibacterial activated carbons prepared from pitch containing organometallics Hisashi Tamai a , Nobuyoshi Katsu a , Kazuhisa Ono b , Hajime Yasuda a , * a
b
Department of Applied Chemistry, Faculty of Engineering, Hiroshima University, Higashi-hiroshima 739 -8527, Japan Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1 -4 -1 Higashi-hiroshima 739 -8527, Japan Received 19 July 2000; accepted 29 December 2000
Abstract A simple method for the preparation of antibacterial activated carbons was reported. The antibacterial activated carbons uniformly contain metal oxides, i.e. CaO, MgO, NiO, CoO and ZnO. These activated carbons were readily prepared by steam activation of pitch containing organometallics, i.e. CaI 2 , t-BuMgBr, Ni(acac) 2 , Co(acac) 3 , and Zn(acac) 2 . The antibacterial activities for B. subtilis, S. aureus, and E. coli were estimated by the growing inhibitory effect in the solutions containing activated carbons and by haloes at surrounding of activated carbons on agar. The CaO, MgO, CoO, and ZnO dispersed activated carbons exhibited obviously the antibacterial activity for B. subtilis both in the solutions and on agar. The MgO and CoO dispersed ones exhibited a growing inhibition effect for S. aureus. On the other hand, no inhibitory effect was observed for the growing of E. coli. 2001 Elsevier Science Ltd. All rights reserved. Keywords: A. Activated carbon, pitch; B. Activation
1. Introduction Activated carbons have attracted much attention as one of the effective adsorbents. For wide applications of activated carbons as adsorbents, the controlling of pore size and its distribution is important. Until a recently there have been many investigations on the controlling of pore size. Recently, we have developed highly mesoporous carbons by activating pitch containing rare earth metal complexes with steam [1–3], and then we showed that metal or metal oxide particles were uniformly dispersed in activated carbons obtained from pitch containing organometallics in terms of electron probe microanalysis (EPMA) and X-ray diffraction (XRD) analyses [3,4]. On the other hand, the antibacterial activity of activated carbons are also very important for application in water purification systems, e.g. purification of drinking water because the growing of bacteria on the surface of activated carbons spoils the function of activated carbon and resulting water may be polluted by bacteria. In general, the antibacterial activities of activated carbons are available as *Corresponding author. Tel.: 181-824-24-7731; fax: 181824-22-7191. E-mail address: [email protected] (H. Yasuda).
Ag deposited activated carbons prepared by impregnation methods [5–7]. However, in the case of Ag supported activated carbons prepared by impregnation or deposition methods, there arise some problems, e.g. needing a few steps for Ag-supporting such as impregnation in Ag solutions and heat-treatment of activated carbons, a decrease in surface area and pore size by deposited Ag, and rapid elution of Ag ions at the initial stage of usage [5]. Sawai et al. [8–10] reported that some metal oxides powders e.g., MgO, CaO, and ZnO showed a growth inhibitory effect for bacteria such as Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis). Based on these results., we expected that metal oxides particles uniformly dispersed in activated carbons should show antibacterial activity. Especially, finely and uniformly dispersing of metal oxide particles in activated carbons plays an important role to realize the excellent antibacterial effect. From these points of view, we attempted the preparation of the antibacterial activated carbons without complicated steps used for the preparation of Ag-supporting activated carbon. We supposed the formation of metal oxides simultaneously with the formation of pores in activated carbons by activating pitch containing organometallics. The obtained activated carbons exhibited antibacterial
0008-6223 / 01 / $ – see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S0008-6223( 01 )00003-3
1964
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
activities for B. subtilis and S. aureus, although their activities were lower than that of Ag deposited activated carbons. We show that the antibacterial activated carbons can be prepared by the simple procedure.
2. Experimental
2.1. Materials Pitch was coal tar pitch from Osaka gas Co. (softening point: 72.28C, C: 93.2%, H: 4.7%, N: 1.1%). CaI 2 , tBuMgBr, Ni(acac) 3 , Fe(acac) 3 , Co(acac) 3 , Al(acac) 3 , and Zn(acac) 2 were purchased from Kanto Chemical and used without further purification. Manganocene (Cp 2 Mn) was synthesized as follows [11]: cyclopentadiene (1.59 mmol) was added slowly to sodium shot (3.65 g) in THF (100 ml), and then to this solution manganous chloride (10 g) was added. The mixture was refluxed for 3 h and THF was removed under vacuum. The resulting Cp 2 Mn was sublimed in vacuo. Dark brown Cp 2 Mn crystals were obtained (yield; 2 g). Tetrahydrofuran (THF) (Kanto Chemical) was dried over Na / K alloy and distilled before use. S. aureus (HUT005), B. subtilis (IFO3134), and Escheritia coli (E. coli) (HUT8106) were obtained from Fermentation Technology Department of Hiroshima University.
2.2. Carbons Various activated carbons were prepared by steam activation of pitch containing organometallics. Pitches containing organometallics were obtained by mixing a THF solution of pitch with THF solutions of organometallics. A typical preparation method is as follows: 4.79 g of pitch was placed in a 300 ml two necked round bottom flask and volatile components were removed under reduced pressure at 1208C. The pitch was dissolved in 90 ml of dry THF. Zn(acac) 2 (1.209 g) dissolved in 90 ml of dry THF was added to the pitch solution. After stirring for 2 h, THF was removed by flash distillation. Activated carbons were obtained by activation of pitch containing organometallics with steam at 9008C. Ag supported activated carbons were prepared by the impregnation method [6]. Activated carbon prepared from organometallics-free pitch by steam activation was impregnated in aq. solutions of AgNO 3 for 24 h. After evaporation of AgNO 3 solution, the activated carbon was heat-treated at 4208C for 1 h.
2.3. Methods Specific surface area and pore size distribution were determined by BET method, and t-plots (for micropore) and BJH [12] (for mesopore) methods, respectively, by applying N 2 adsorption isotherms measured using a Quantachrome Autosorb-6. XRD analyses were performed using
a Rigaku RDA-1B system with Cu Ka radiation. The dispersion of metal oxides in activated carbons was observed by electron probe microanalysis (EPMA) with a JEOL JXA-8900. The metal contents in carbons were measured by particle-induced X-ray emission (PIXE) analysis and ash contents in activated carbons. PIXE analysis was performed using a van de Graaff accelerator of Hiroshima University. The intensity of the characteristic X-ray generated by irradiation of H 1 was measured. We estimated antibacterial activities of metal oxides particles dispersed in activated carbons by the growing inhibitory effect for B. subtilis, S. aureus, and E. coli. A growing inhibitory effect was determined by two kinds of methods; (1) measurement of bacteria growing in the solutions containing activated carbons as follows: a portion of B. subtilis was inoculated in 5 ml of sporulation medium (beef extract 10 g, poly-pepton 10 g, NaCl 5 g / 1 l of pure water). The medium was incubated at 378C for 18 h. The spores were inoculated in 5 ml of sporulation medium and to this medium activated carbons were added. The spores were filtered after the prescribed time. The filtrate (0.5 ml) was diluted by 4.5 ml of sporulation medium. The bacteria growing was determined by measuring optical density of the diluted solution. Optical density (OD 660 ( l 5660 nm)) was measured by UV–Visible spectrometer(Pharamacia LKB Biochrome 4060 UV–Visible Spectrometer). (2) Measurement by the halo method as follows [12]: bacteria was inoculated in 5 ml of a sporulation medium (poly-pepton 10 g, yeast extract 2 g, and magnesium sulfate 1 g / 1 l of pure water), and this solution was incubated at 378C for 18 h. The spores were inoculated in 100 ml of sporulation medium and incubated at 378C for 18 h. Agar containing these spores was placed onto agar plates containing activated carbons and dried for a few minutes. After incubating for 24 h at 378C, the lengths of haloes formed surrounding the activated carbons were measured.
3. Results and discussion Table 1 shows the carbonized yields and pore characteristics of the activated carbons obtained from pitches containing various organometallics. The addition of CaI 2 lowered the yield and BET specific surface area. The formed CaO accelerated the consumption of carbon precursors such as pitch during steam activation and as a result, the formed pores were supposed to be destroyed throughout activation. On the other hand, the addition of other organometallics, i.e. t-BuMgBr, Zn(acac) 2 , Ni(acac) 3 , Fe(acac) 3 , Co(acac) 3 , and Al(acac) 3 into pitch promotes the formation of pores and BET specific surface areas are higher than that from organometallics-free pitch in spite of the short activation time. Especially, the promoting effect for pore formation is remarkable in the addition of
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
1965
Table 1 Preparation of activated carbons from pitches containing various organometallics a Sample
Organometallics
Metal in pitch (wt.%)
Activation time (min)
Yield (%)
BET surface area (m 2 / g)
Mean pore size (nm)
Ca–AC Mg–AC Zn–AC Mn–AC Ni–AC Fe–AC Co–AC Al–AC AC
CaI 2 t-BuMgBr Zn(acac)2 Cp 2 Mn Ni(acac) 2 Fe(acac) 3 Co(acac) 3 Al(acac) 3 –
5.0 5.0 5.0 2.5 5.0 5.0 5.0 5.0 –
3 15 15 25 12 15 18 18 30
14.8 40.2 26.3 31.8 28.2 36.9 24.6 32.0 24.7
0.4 118 150 76 131 159 159 221 88
50.0 3.1 2.6 3.6 3.2 3.8 3.8 2.2 2.5
a
Activation temp.: 9008C.
Co(acac) 3 and Al(acac) 3 . Fig. 1 shows the pore distributions of activated carbons obtained from pitches containing t-BuMgBr and Zn(acac) 2 . Micropores of about 0.5 nm of pore radius were mainly developed in activated carbon obtained from pitch containing t-BuMgBr, whereas pores of large size (mesopores) were formed in addition to micropores by the addition of Zn(acac) 2 . Similarly, a large ratio of micropores and small ratio of mesopores were formed in activated carbons obtained from pitches containing other organometallics. The decomposition of organometallics results in the formation of metal or metal oxide in the course of activation of pitch [2,4]. We measured metal contents in the activated carbons obtained. The results are shown in Table 2. Metal contents in the activated carbons range from 4.3 to 9.8 wt.% in the cases of Zn, Ni, Fe, Co, and Ni. These values were lower compared with the theoretical values calculated from metal contents in pitch (5 wt.%). A part of metal atoms is supposed to disappear throughout the decomposition of organometallics during activation. On the other hand, the detection of light atoms such as Ca
and Mg by PIXE analysis was impossible. Based on metal contents determined from ash contents in activated carbons obtained from pitches containing CaI 2 and t-BuMgBr, it is supposed that most metal atoms remain in the activated carbons. In addition, XRD analysis indicated the formation of crystals of CaO and MgO in the activated carbons as described below. Sawai et al. [8–10] reported that some metal oxides showed antibacterial activities. We analyzed metal species formed from organometallics in activated carbons during activation by XRD analyses. Fig. 2 shows XRD patterns of the activated carbons obtained from pitches containing CaI 2, t-BuMgBr, Zn(acac) 2 , and Cp 2 Mn. The peaks due to CaO, MgO, and ZnO on each XRD patterns were observed in addition to the broad peak of carbon crystallites. As shown in Fig. 2, MnO and Mn 2 O 3 particles were formed from Cp 2 Mn. Similarly, the formation of metal oxides, i.e. Al 2 O 3 , and Fe 2 O 3 from Al(acac) 3 and Fe(acac) 3 , respectively, was observed. On the other hand, the formation of metal particles, i.e. Co and Ni in addition to metal oxides such as CoO and NiO from Co(acac) 3 and Ni(acac) 3 , was observed. Table 2 Metal content in activated carbons obtained from pitches containing organometallics
Fig. 1. Pore distributions of activated carbons obtained from pitches containing t-BuMgBr and Zn(acac) 2 (s); t-BuMgBr, (d); Zn(acac) 2 .
Sample
Metal in pitch (wt.%)
Metal in carbon (wt.%)
Ca–AC Mg–AC Zn–AC Mn–AC Ni–AC Fe–AC Co–AC Al–AC
5.0 5.0 5.0 2.5 5.0 5.0 5.0 5.0
25.0 a 9.9 a 4.4 b 0.8 b 9.8 b 6.5 b 8.6 b 4.3 b
a b
Determined from ash content. Determined by PIXE.
1966
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
Fig. 2. XRD patterns of activated carbons obtained from pitches containing organometallics (a), CaI 2 ; (b), t-BuMgBr; (c), Zn(acac) 2 ; (d), Cp 2 Mn.
Fig. 4. Optical densities of sporulation medium containing Ca– AC for B. subtilis. Concentration of activated carbon (d); Ca–AC 10 mg / ml, (m); Ca–AC 30 mg / ml, (n); AC 30 mg / ml, (s); none.
The dispersion states of metal oxides in the activated carbons obtained from pitches containing organometallics are important for antibacterial activity of activated carbons. The homogeneous dispersion of metal oxides were observed by EPMA. SEM and EPMA images of the activated carbon obtained from pitch containing Zn(acac) 2 are exemplified in Fig. 3. ZnO were dispersed almost uniformly in activated carbon. The distributions of other metal oxides were also uniform as observed for ZnO. The antibacterial activities for B. subtilis, S. aureus, and
E. coli were investigated. Bacteria were incubated in sporulation medium containing activated carbons and the change of optical density (OD) of medium with incubation time was measured. Figs. 4–7 show the results on the activated carbons obtained from pitches containing CaI 2 , t-BuMgBr, Zn(acac) 2 , and Cp 2 Mn, for B. subtilis. As shown in Figs. 4 and 5, in the cases of the activated carbons obtained from pitches containing CaI 2 and tBuMgBr, no increase of OD with time was observed while
Fig. 3. EPMA image of activated carbon obtained from pitch containing Zn(acac) 2 .
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
Fig. 5. Optical densities of sporulation medium containing Ma– AC for B. subtilis. Concentration of activated carbon (d); Mg– AC 10 mg / ml, (m); Mg–AC 30 mg / ml, (j); Mg–AC 50 mg / ml, (n); AC 30 mg / ml, (s); none.
the OD of medium without activated carbon or the OD of medium containing activated carbon obtained from organometallics-free pitch increased with incubation time. These results obviously indicate that the activated carbons obtained from pitches containing CaI 2 and t-BuMgBr possess strong antibacterial activities for B. subtilis. In addition, the activated carbons obtained from pitches containing Co(acac) 3 and Ni(acac) 3 showed the same antibacterial effect for B. subtilis as that from pitch containing t-BuMgBr. As shown in Fig. 6, the OD of sporulation medium for activated carbon containing ZnO slightly increases at the initial stage of incubation and the value of OD decreased with increasing the amount of
Fig. 6. Optical densities of sporulation medium containing Zn– AC for B. subtilis. Concentration of activated carbon (m); Zn–AC 30 mg / ml, (d); Zn–AC 50 mg / ml, (n); AC 30 mg / ml, (s); none.
1967
Fig. 7. Optical densities of sporulation medium containing Mn– AC for B. subtilis. Concentration of activated carbon (d); Mn– AC 30 mg / ml, (m); Mn–AC 50 mg / ml, (n); AC 30 mg / ml, (s); none.
activated carbon added in incubation medium. The ZnO dispersed activated carbon is supposed to suppress growing of bacteria, although the antibacterial activity was lower than that of the MgO dispersed one. On the other hand, as shown in Fig. 7, the activated carbon containing MnO and Mn 2 O 3 exhibited no growing inhibitory effect for B. subtilis. Fig. 8 shows the changes of ODs of sporulation medium containing activated carbons for S. aureus with incubation time. Mediums containing activated carbons prepared from pitch containing Zn(acac) 2 or metal oxide-free activated carbon exhibited the same growing curves as that in medium without activated carbon. There is no antibacterial activity in activated carbon containing ZnO for S. aureus.
Fig. 8. Optical densities of sporulation medium containing 30 mg / ml of activated carbons for S. aureus (j); Ca–AC, (s); Mg–AC, (n); Zn–AC, (h); Ni–AC, (m); AC, (d); none.
H. Tamai et al. / Carbon 39 (2001) 1963 – 1969
1968
Fig. 9. Optical densities of sporulation medium containing 30 mg / ml of activated carbons for E. coli (j); Ca–AC, (s); Mg– AC, (n); Zn–AC, (h); Ni–AC, (앳); Co–AC, (m); AC, (d); none.
On the other hand, the increase in ODs of mediums containing the NiO, MgO, and CaO dispersed activated carbons is low, compared with the OD of medium containing metal oxide-free activated carbon. These activated carbons exhibited an inhibitory effect for the growing of S. aureus. On the antibacterial activity for E. coli, Fig. 9 shows the relation between ODs of sporulation mediums containing activated carbons and incubation time. ODs of mediums containing metal oxides dispersed activated carbons scarcely increased with time. However, the OD of medium containing metal oxide-free activated carbons was also low. The growing inhibitory effect of the metal oxides dispersed activated carbons for E. coli seems due to the adsorption of E. coli on activated carbons rather than the antibacterial activity of metal oxides. Furthermore, in order to clarify the antibacterial activity of metal oxides dispersed activated carbons, we measured the growing inhibitory effect on agar by the halo method. Table 3 shows the lengths of haloes formed surrounding the activated carbons. The activated carbons containing
Table 3 Growth inhibitory effect for bacteria on agar Sample
Ca–AC Mg–AC Zn–AC Ni–Ac Fe–AC Co–AC Al–AC AC–Ag5 AC–Ag10
CaO, MgO, ZnO, and CoO show the formation of haloes of almost the same lengths for B. subtilis. This formation of haloes indicates that these activated carbons give the inhibitory effect for the growth of B. subtilis. However, the lengths of haloes are half of that by Ag supported activated carbons, which were prepared by the impregnation method using aq. solution of AgNO 3 . On the other hand, NiO dispersed activated carbon for B. subtilis and S. aureus and CaO dispersed activated carbon for S. aureus, no growing inhibitory effect was observed on agar in spite of the fact that it exhibited the antibacterial activity in the solutions. This may be due to the difference in contact area of activated carbons to bacteria. The contact area of activated carbon in solutions is supposed to be larger than that on agar. These results suggest that the antibacterial activity of NiO dispersed activated carbon is not so strong as that of MgO dispersed activated carbon.The activated carbons obtained from pitches containing organometallics showed no inhibitory effect on agar for E. coli, in spite of that Ag supported activated carbon exhibited strong inhibitory effect.
4. Conclusion The antibacterial activated carbons can be simply prepared by steam activation of pitch containing organometallics. Metal oxides were formed uniformly in activated carbons and the antibacterial activities were dependent on the kind of metal oxides. The CaO, MgO, CoO, and ZnO dispersed activated carbons obtained from pitches containing CaI 2 , t-BuMgBr, Co(acac) 3 , Ni(acac) 2 , and Zn(acac) 3 , respectively, exhibited good antibacterial activities for B. subtilis. The MgO and CoO dispersed ones presented a growing inhibitory effect in sporulation solutions and on agar for S. aureus. On the other hand, no inhibitory effect was observed for the growing of E. coli. The antibacterial activated carbons do not need the complicated procedures such as that for the preparation of Agsupporting activated carbon.
Acknowledgements
Halo length (mm) B. subtilis
E. coli
S. aureus
4.2 4.0 4.2 0 0 4.5 0 9.9 9.9
0 0 0 0 0 0 0 4.0 6.0
0 8.5 0 0 0 6.5 0 9.0 13.3
This work is partially supported by a Grant-in-aid for ‘Research for the Future Program’, Nano-carbon, from the Japan Society for the Promotion of Science.
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