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Effective factor on antibacterial characteristics of Mg1−XNiXO solid solution
chemical engineering research and design 9 1 ( 2 0 1 3 ) 1055–1062
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Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd
Effective factor on antibacterial characteristics of Mg1−X NiX O solid solution Toshiaki Ohira a,∗ , Osamu Yamamoto b a
Center for Geo-Environmental Science, Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata Gakuen-machi, Akita 010-8502, Japan b Department of Bio-System Engineering, Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa-shi, Yamagata 992-8510, Japan
a b s t r a c t Mg1−X NiX O solid solution powder samples with different chemical compositions were prepared by heating MgO–NiO mixtures at 1300 ◦ C for 12 h in air. From XRD measurement, all powder samples were indexed as a single phase of cubic structure, of which the diffraction peaks shifted to high-angle side with the increase of doping amount of NiO. The pH values of the solution dispersed with the powder samples decreased when the doping amount of NiO in solid solution was increased. Antibacterial activity of the powder samples was examined by colony count method. In the result, the antibacterial activity of Mg1−X NiX O was remarkably weaker than original MgO powders, irrespective of the kind of bacteria. In addition, it was found that the antibacterial activity of Mg1−X NiX O reduced with increasing the doping amount of NiO. Two factors, the generated amount of O2 − and the eluted amount of Ni2+ ions affected the antibacterial activity of Mg1−X NiX O solid solution. Especially, the stability of O2 − in aqueous solution is dependent on pH value. Therefore, the strength of antibacterial activity was associated with the pH values in the dispersed solution of Mg1−X NiX O. © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: MgO; NiO; Solid solution; Antibacterial activity; Colony count
1.
Introduction
Recently, magnesium oxide (MgO) has attracted strong interest as an antibacterial ceramic, because it has shown antibacterial activity in only small amounts of powder without the presence of light (Sawai et al., 1997a; Tamai et al., 2001). Sawai et al. (1997b) reported that main factors contributing to the occurrence of antibacterial activity were assumed to be the increase of the pH value because of the hydration of MgO, and super-oxide (O2 − ) generated from the surface of MgO. And they found that such powder characteristics of MgO as particle size and powder concentration, affected the antibacterial activity toward Escherichia coli and Staphylococcus aureus (Sawai et al., 1996a). Furthermore, extensive research on the antibacterial activity of MgO has shown that cubictype solid solution in the MgO–ZnO system exhibited stronger antibacterial activity, when compared with the original MgO (Ohira et al., 2008). In detail, antibacterial activity of cubic-type
∗
Mg1−X ZnX O solid solution that is enhanced with an increased doping amount of ZnO, is probably caused by the increase in the generated amount of O2 − . Therefore, it is important to understand the effective factors of the solid solutions with MgO as one of the end members on antibacterial activity, in order to know the antibacterial mechanism of the Mg1−X MX O (with M = Zn, Ni, etc.) solid solutions. Except for this understanding of Mg1−X ZnX O solid solution, however, there are no studies on the antibacterial activity of other Mg1−X MX O solid solutions having a cubic structure. According to a phase diagram of the MgO–NiO system, MgO and NiO can form complete solid solution having a cubic structure (Liu et al., 1964). Since antibacterial activity of Mg1−X NiX O solid solution may be dependent on the doping amount of NiO, a solid solution with different chemical compositions was prepared by heating the mixtures of MgO and NiO at 1300 ◦ C for 12 h. Using the colony count method, the antibacterial activity of Mg1−X NiX O solid solution having different chemical
Corresponding author. Tel.: +81 18 889 2450; fax: +81 18 837 0409. E-mail address: [email protected] (T. Ohira). Received 9 August 2012; Received in revised form 19 October 2012; Accepted 25 November 2012 0263-8762/$ – see front matter © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cherd.2012.11.015
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Table 1 – Sample code, molar ratio and chemical composition of powder samples used in this study. Sample code
Molar ratio (MgO/NiO)
MN05 MN10 MN20 MN50 MN80
19.0 9.00 4.00 1.00 0.25
Chemical composition Mg0.95 Ni0.05 O Mg0.90 Ni0.10 O Mg0.80 Ni0.20 O Mg0.50 Ni0.50 O Mg0.20 Ni0.80 O
compositions was evaluated, with an emphasis on the effect of the chemical composition on the solid solution.
concentration of 0.63 g dm−3 were independently collected by filtration, and dried at 100 ◦ C for 12 h. The crystal phases of the dried powders were evaluated by XRD, in order to confirm the formation phase of Mg1−X NiX O after hydration, because Mg(OH)2 and Ni(OH)2 may be formed by the hydration of MgO and NiO, and this can be detected by XRD. Thus, the degree of hydration of the powder samples was evaluated. Inductively coupled plasma atomic emission spectrometry measurement (ICP-AES; Seiko Instruments, Inc., SPS 7700) was performed to measure the amount of Ni2+ ions eluted from the solid solution. This was done because the Ni2+ ions are known to show antibacterial activity (Sönmez et al., 2006).
2.
Materials and methods
2.3.
2.1.
Preparation of materials
Escherichia coli 745 (hereafter, E. coli), a Gram-negative bacterium, and Staphylococcus aureus 9779 (hereafter, S. aureus), a Gram-positive bacterium, were used in antibacterial tests. These test bacteria were cultured aerobically in LB medium containing 0.5% yeast extract (Becton, Dickinson and Co.), 1% bactopeptone (Becton, Dickinson and Co.), and 1% sodium chloride (NaCl; Wako Pure Chemical Ind., Ltd., purity: 99.9%) at 36 ◦ C for 48 h. Bacteria suspended in the medium were cultured at 36 ◦ C for 48 h on a reciprocal shaker, and rinsed four times with sterile water. Finally, the bacterial suspension was diluted with sterile water at a concentration of 107 cfu dm−3 (cfu: colony forming unit).
The sample code, molar ratio (MgO/NiO) and chemical composition of Mg1−X NiX O solid solution are summarized in Table 1. The number in the sample code represents the molar fraction of NiO. For example, MN20 represents the 80 mol%MgO–20 mol%NiO powder sample. The powder samples of Mg1−X NiX O solid solution were prepared using a solid-state reaction. The mixtures of MgO (Ube Chemical Ind, Ltd., purity: 99.9%) and NiO (Nacalai Tesque, Inc., purity: 97.0%) were heated at 1300 ◦ C for 12 h in air. As reference materials against the as-prepared powder samples, pure MgO and NiO powders were prepared under identical heat-treatment conditions. In this work, MgO–NiO mixtures were heated at 1300 ◦ C, which is below both the melting and boiling points for NiO and MgO. Stoichiometrically, therefore, chemical compositions based on Mg1−X NiX O (X = 0.05, 0.10, 0.20, 0.50 and 0.80), should be unchanged with the mixed ratio of MgO–NiO mixtures.
2.2.
Powder characterization
The formation phase of the powder samples was identified by X-ray diffraction measurement (XRD; Rigaku, RAD-C SYSTEM). The scanning rate was 1◦ /min from 2 = 20◦ to 80◦ and the operation voltage and current of XRD were 40 kV and 20 mA, respectively. The value of the lattice constant was calculated using the diffraction peaks detected on XRD. The lattice constants (a0 ) of MgO, NiO and five Mg1−X NiX O powders were calculated by Eq. (1): a0 = {(h2 + k2 + l2 ) × d2 }
1/2
(1)
where h, k, and l are Miller indices and d is the interplane distance in nm. The specific surface area of the powder samples was determined by measuring the adsorption isotherms of N2 at −196 ◦ C (BET; Beljapan, Inc., BELSORP-mini), because of one of the powder characteristics affecting antibacterial activity (Yamamoto et al., 1998). Since the pH value in a bacterial growth environment is known to affect antibacterial activity (Small et al., 1994), it is essential to measure the pH value of the solution dispersed with the powder samples. Powder samples were dispersed into distilled water at a concentration of 0.63 and 40 g dm−3 to make powder slurries. After keeping the dispersed solution for 1 h at 36 ◦ C, the pH value of the solution was measured. After pH measurement, the powder slurries with a
2.4.
Preparation of bacterial suspension
Antibacterial test
The bacterial suspension was added into sterile water containing powder samples at a concentration of 0.63–40 g dm−3 . Thus, the antibacterial test was started, and each suspension was kept at 36 ◦ C for different amounts of time (15, 30, 45 and 60 min) on a reciprocal shaker. A 10−4 dm3 suspension pipetted at specified times (15, 30, 45 and 60 min) was spread on a nutrient agar (NA, Eiken Chemical, Co.) against E. coli, and spread on a pearl-core plate count agar (PPCA, Eiken Chemical, Co.) against S. aureus. These agars were incubated at 36 ◦ C for 24 h without the presence of light. After incubation, the colonies formed on the agar were counted. In vitro antibacterial activity of the powder samples was assessed by calculating the survival ratio = N/N0 , where N and N0 are, respectively, the viable bacterial counts N (cfu dm−3 ) at a specified time, and the initial counts of bacteria N0 (cfu dm−3 ) (Seven et al., 2004).
2.5.
Antibacterial activity of Ni2+ ions
The prepared bacterial suspension (Section 2.3) was added into nickel acetate solution with a concentration of 50 × 10−5 mol dm−3 , and an antibacterial test using nickel acetate solution was started. After 15, 30, 45 and 60 min, a 10−4 dm3 solution was adopted, and spread on either a nutrient agar against E. coli, or on a pearl-core plate count agar against S. aureus. After incubating these agars at 36 ◦ C for 24 h without the presence of light, the colonies formed on the agar were counted. By means of the calculation described in Section 2.4, the survival ratio was estimated. Based on the degree of the decline of the survival ratio on E. coli and S. aureus, the sensitivity to Ni2+ ions was compared between E. coli and S. aureus.
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Intensity/Arb.Unit
(g) (f) (e) (d) (c)
8 7 6
5 4 3 2 1 0
(222)
(311)
(220)
(111)
(200)
(b)
(a) 20
30
40
50
60
70
Fig. 1 – XRD patterns of powder samples obtained by heating at 1300 ◦ C for 12 h in air: (a) MgO, (b) MN05, (c) MN10, (d) MN20, (e) MN50, (f) MN80 and (g) NiO.
3.
Results and discussion
3.1.
Powder characterization
Fig. 1 shows XRD patterns of the powder samples obtained by heating at 1300 ◦ C, together with the pure powders NiO and MgO, both obtained by identical heat-treatment. A single phase of a cubic structure was formed in all obtained powder samples. The diffraction peaks corresponding to MgO were very similar to those of NiO, and shifted to the high-angle side when the doping amount of NiO was increased. The value of the lattice constant on the powder samples was calculated using the 200 diffraction peaks, which were consistent with the values estimated from Vegard’s rule. As shown in Fig. 2, the lattice constant was found to decrease linearly with the increase of the doping amount of NiO. This is due to the fact that the ionic radius of the Mg2+ ion is larger than that of the Ni2+ ion. Indeed, the ionic radii of the Mg2+ and Ni2+ ions were 72 pm and 69 pm, respectively. Thus, the decrease in the lattice constant seems to be attributed to the substitution of Mg2+ 0.4215 0.4210 0.4205 0.4200 0.4195 0.4190 0.4185 0.4180 0.4175 0.00
0.20
0.40
0.60
0.80
1.00
x in Mg1-XNiXO Fig. 2 – Change in lattice constant of MgO.
0
0.5
1
Relative pressure, p/p0 / Fig. 3 – N2 adsorption isotherms at −196 ◦ C on powder samples. 䊉: MgO, : MN05, : MN10, : MN20 and ♦: MN50.
80
Diffraction Angle, 2θθ / ° (CuKα α )
Lattice constant / nm
9 Volume absorbed / cm3 g-1
(222)
(311)
(220)
(200)
(111)
10
:MgO(cubic) :NiO(cubic)
1.20
ions by Ni2+ ions. These results indicate that Mg1−X NiX O solid solutions (MN05, MN10, MN20, MN50 and MN80) were successfully formed, which are consistent with various reports on the Mg1−X NiX O solid solution (Hu and Ruckenstein, 1996; Nurunnabi et al., 2005; Rao and Sunandana, 2008). NiO is known to start melting at 1980 ◦ C at 1 atm. Thus, any heattreatment above 1980 ◦ C may be able to change the expected compositions based on Mg1−X NiX O stoichiometrically. Since the heating temperature performed in this study was 1300 ◦ C, the MgO/NiO ratio in the obtained Mg1−X NiX O solid solutions should be equal to the ratio in the MgO–NiO mixtures. Yamamoto et al. (1998) have reported antibacterial activity of ZnO enhanced with an increase in the specific surface area; this, because of the increase in the generated amount of H2 O2 on its surface. That is, the specific surface area of the powder samples is one of the powder characteristics affecting antibacterial activity. Therefore, measuring the specific surface area of powder samples is important for precisely evaluating antibacterial activity. By BET measurement, the specific surface area of the obtained five solid solutions was below 1 m2 g−1 , having no accuracy for quantitative analysis. Fig. 3 shows the obtained N2 adsorption isotherms at −196 ◦ C of pure MgO, MN05, MN10, MN20 and MN50. It was found that the amount of N2 adsorbed on MgO was much larger than that on Mg1−X NiX O. On the other hand, there was little difference in the amount of the adsorbed N2 in Mg1−X NiX O. From these results, the specific surface areas of Mg1−X NiX O powder samples were judged to be very similar. It has been reported that increasing the pH value causes the enhancement of antibacterial activity (Sawai et al., 2001). Thus, measuring the pH value in aqueous solution containing the powder samples is essential to evaluate antibacterial activity. As summarized in Table 2, the pH values of the powder samples decreased when the doping amount of NiO in solid solution was increased. Fig. 4 shows XRD patterns of the dried powders after pH measurement. In the case of the original MgO, diffraction peaks corresponding to Mg(OH)2 of a hexagonal type were detected. On the other hand, no peaks of Ni(OH)2 were detected in any of the powder samples. These results indicate that the degree of hydration in the original MgO was the highest for all powder samples. Therefore, the reason that original MgO showed the highest pH value might be assumed to be due to the formation of Mg(OH)2 resulting from the hydration
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Table 2 – Sample code, specific surface areas, pH values and eluted Ni2+ ion concentration of powder samples in this study. Specific surface area (m2 g−1 )
Sample code
0.63 g dm−3 MgO MN05 MN10 MN20 MN50 MN80 NiO
8.4 <1 <1 <1 <1 <1 <1
11 8.8 8.0 7.5 6.2 5.5 5.5
Intensity/Arb.Unit
of MgO. On the other hand, there were no diffraction peaks in either Mg(OH)2 or Ni(OH)2 in MN05, MN10, MN20, MN50 and MN80. This may mean that the formation of Mg1−X NiX O solid solution inhibits hydration. Therefore, the inhibition of hydration might be related to the decrease of pH values in the Mg1−X NiX O powder samples. This implies that the effect of pH values on the antibacterial activity became weak as the doping amount of NiO was increased. Chandra et al. (2008) have suggested that Ni2+ ions show only slight antibacterial activity. Therefore, the amount of Ni2+ ions eluted from the powder samples was measured by ICP-AES. The amount of Ni2+ ions eluted from the powder samples increased, reaching 50% of the doping amount of NiO. It was also found that the amount of eluted Ni2+ ions decreased. Regarding NiO itself, the amount of eluted Ni2+ ions was the smallest of all the powder samples. It has been reported that the MgO content in solid solution affects the degree of hydration (Katyal et al., 1998). In our comparison of pH values between the powder concentrations of 0.63 and 40 g dm−3 , similar values were observed in both MN80 and NiO. That is, pH values of dispersions with the powder concentration of 0.63 and 40 g dm−3 were 5.5 and 5.7, respectively.
20
40
60
80
Diffraction Angle, 2θθ / ° (CuKα α ) Fig. 4 – XRD patterns of powder samples after dispersed into distilled water at a concentration of 0.63 g dm−3 .
Eluted Ni2+ ion concentration (mol dm−3 )
pH value 40 g dm−3 – 9.7 8.7 8.4 6.8 5.7 5.7
– 5.1 × 10−5 12 × 10−5 13 × 10−5 18 × 10−5 8.7 × 10−5 3.3 × 10−5
On the other hand, the pH values of MN05, MN10, MN20 and MN50 significantly increased when the powder concentration was increased. This result seems to indicate a higher hydration resistance for MN80 and NiO. If the hydration resistance of Mg1−X NiX O solid solution is high, the solubility should be low. Therefore, the reason that the eluted amount of Ni2+ ions decreased in MN80 and NiO might be presumed to be due to their high hydration resistance.
3.2.
Antibacterial activity
The relative strength of antibacterial activity on Mg1−X NiX O solid solution having different chemical compositions was evaluated by the colony count method. When the survival ratio changes with a steep decrease at a specified time, the colony count method helps us see strong antibacterial activity in powder samples. Fig. 5(a) and (b) shows the survival ratio of S. aureus and E. coli, respectively, using pure MgO and NiO powders suspended at the powder concentration of 0.63 g dm−3 . In both S. aureus and E. coli, the reduction of the survival ratio on MgO was much larger than that on NiO. That is, MgO showed antibacterial activity, while no antibacterial activity was recognized in NiO. Fig. 6(a) and (b) shows the survival ratios of S. aureus and E. coli, respectively, using five solid solutions suspended at the powder concentration of 0.63 g dm−3 . In S. aureus, the values of the survival ratio on MN05, MN10 and MN20 decreased over time. There was no significant decrease observed in either MN50 or MN80. In a comparison of the survival ratios at specified times for MN05, MN10 and MN20, the survival ratio was found to decrease when the doping amount of NiO in solid solution was increased, although the behavior resulting from the reduction of MN05 was similar to that of MN10. In the case of E. coli, the reduction behavior of the survival ratio was almost identical with that of S. aureus. That is, the antibacterial activity toward E. coli was found to be less with an increased amount of NiO doping. MN50 and MN80 did not show any significant antibacterial activity. Regarding the antibacterial activity of MgO, it is reported that the generation of O2 − , as an antibacterial agent, was detected on its surface (Sawai et al., 1996b). In the case of the Mg1−X NiX O solid solution, the O2 − generated from the surface of the solid solution, therefore, might be a chemical species contributing to the occurrence of antibacterial activity. Antibacterial activity should be enhanced with an increase of the specific surface area of powder samples because of the increase in the amount of active oxygen such as O2 − and H2 O2 generated on its surface. As described in Section 3.1, the specific surface area values of Mg1−X NiX O solid solutions were of similar. This implies that the effect of specific surface areas on antibacterial activity is probably also similar. However, antibacterial activity
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Fig. 5 – Survival ratio of (a) S. aureus and (b) E. coli on MgO and NiO powder samples slurries at powder concentration of 0.63 g dm−3 . : MgO and 䊉: NiO. of the five solid solutions was less when the doping amount of NiO was increased. These results indicate that there is an effective factor contributing to the occurrence of antibacterial activity besides the specific surface area. In the comparison of the survival ratios at specified times between pure MgO and the five solid solutions (see Figs. 5 and 6), the decrease of the survival ratio on the five solid solutions is smaller than that of pure MgO. This means that the antibacterial activity of Mg1−X NiX O solid solution was weaker than that of pure MgO. Chang et al. (2008) reported that the culturability of Legionella pneumophila decreased when the pH values increased in the range from 6 to 10. It has also been reported that the effect of pH values on antibacterial activity was remarkably enhanced when pH values were greater than 10 (Small et al., 1994; Sawai et al., 2001). The pH value of MgO was 11 in the present study, and showed the strongest antibacterial activity of all powder samples. When increasing the doping amount of NiO, the pH values of the five solid solutions decreased. Similarly, the antibacterial activity on the five solid solutions decreased when the doping amount of NiO was increased, although MN50 and MN80 did not show significant antibacterial activity. From these results, it is reasonable
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Fig. 6 – Survival ratio of (a) S. aureus and (b) E. coli on solid solution slurries at powder concentration of 0.63 g dm−3 . : MN05, : MN10, ♦: MN20, : MN50 and ×: MN80. to believe that a decrease of pH values is related to a reduction of antibacterial activity when the doping amount of NiO is increased. That is to say, the antibacterial activity of the powder samples might be dependent on the pH values. Figs. 7 and 8 show changes in the survival ratio of S. aureus and E. coli having different powder concentrations, using MN05 and MN20. The survival ratio was found to decrease when the powder concentration increased, irrespective of the kind of bacteria, indicating that the antibacterial activity of MN05 and MN20 was enhanced with an increase of powder concentration. As described in Table 2, the pH values of five solid solutions were below 10; this is known to be inadequate to exhibit antibacterial activity for S. aureus and E. coli (Small et al., 1994; Sawai et al., 2001). This result implies that the generated amount of chemical species that contributes to the occurrence of antibacterial activity increased with the increase of powder concentration. As described above, the chemical species contributing the occurrence of antibacterial activity in the Mg1−X NiX O solid solution should be O2 − generated from the surface of the solid solution. It has been reported that a self-dismutation reaction of O2 − proceeds rapidly when the pH value in an aqueous solution decreases from 10 to 7
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Fig. 7 – Survival ratio of S. aureus on (a) MN05 and (b) MN20 slurries at each powder concentration. : 0.63 g dm−3 , : 2.5 g dm−3 , :10 g dm−3 and 䊉: 40 g dm−3 . (Riley et al., 1991). As expressed by the following equation (2), the self-dismutation reaction of O2 − results in the formation of hydro peroxide (H2 O2 ). And H2 O2 is also known to show antibacterial activity toward E. coli and S. aureus (Mazzola et al., 2009). 2O2 − + 2H+ → H2 O2 + O2
(2)
Since the pH value of the dispersed solution of MN20 was lower than that of MN05, the self-dismutation reaction of O2 − on MN20 might be faster than that on MN05. In other words, O2 − in the dispersed solution of MN05 was more stable, when compared to that of MN20. Assuming that H2 O2 generated by the self-dismutation reaction of O2 − contributed to antibacterial activity, the antibacterial effect of H2 O2 might be less than that of O2 − , depending on the amount of O2 − . Thus, the reason for the enhancement of antibacterial activity with an increased powder concentration may be associated with the stability of O2 − , in addition to the increased generated amount of O2 − on the surface. Generally, the death of bacteria has been reported to be logarithmic in order (Rahn, 1929). When the survival ratios decrease linearly (Figs. 7 and 8), the death rate of bacteria can
Fig. 8 – Survival ratio of E. coli on (a) MN05 and (b) MN20 slurries at each powder concentration. : 0.63 g dm−3 , : 2.5 g dm−3 , :10 g dm−3 and 䊉: 40 g dm−3 . be understood by first-order kinetics as dN/dt = −kN: where t, N and k are, respectively, time, survival ratio, and the death rate constant (Sawai et al., 2005). Thus, a high k value means strong antibacterial activity. Fig. 9(a) and (b) shows the relationship between k values and powder concentrations toward S. aureus and E. coli, respectively. The k values of MN05 were always higher than those of MN20 in the range of powder concentration performed in this study, irrespective of the kind of bacteria; that is, the antibacterial action of MN05 was stronger than that of MN20. In Fig. 9(a), the slope showing S. aureus has the identical value of 0.65 for both MN05 and MN20. In the case of E. coli (see. Fig. 9(b)), the slope degree of MN20 is higher than that of MN05. The slope of the line indicates the sensitivity to chemical species that contribute to antibacterial activity. It is believed that the difference of the slope degree reflects changes in sensitivity to the eluted Ni2+ ions irrespective of the amount of generated O2 − , because the amount of Ni2+ ions eluted from MN20 was found to be greater than that eluted from MN05. Fig. 10 shows the survival ratios of S. aureus and E. coli when using nickel acetate at a concentration of 50 × 10−5 mol dm−3 . In comparing the survival ratios of S. aureus and E. coli, the ratio of S. aureus was less than that of E. coli; that is, the antibacterial
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Fig. 9 – Relationship between powder concentration and death rate constant (k) for (a) S. aureus and (b) E. coli. : MN05 and ; MN20.
action of the Ni2+ ion toward E. coli was stronger than it was toward S. aureus. Acetate salts such as zinc acetate and potassium acetate have been known as a chemical species during the occurrence of antibacterial activity (Atmaca et al., 1998; Lee Yee-lean Cesario et al., 2002; Roe et al., 2002). In these reports, an acetate concentration above 230 × 10−5 mol dm−3 for zinc acetate and 130,000 × 10−5 mol dm−3 for potassium acetate showed antibacterial activity toward S. aureus. In addition to these reports, it has been revealed that acetic acid with a concentration of 830 × 10−5 mol dm−3 has no bactericidal effect (Saene et al., 1985). Since these concentrations were remarkably higher than 50 × 10−5 mol dm−3 , the effect of the acetate ion on antibacterial activity can be ignored for those antibacterial tests in our study where nickel acetate was used. In Fig. 10, therefore, the occurrence of antibacterial activity was presumed to be due to Ni2+ ions. And the antibacterial action of the Ni2+ ion toward E. coli as Gram-negative bacteria, was stronger than it was toward S. aureus as Grampositive bacteria. Consequently, the reason that the slope degree when using E. coli on MN20 was higher than when used on MN05, may be assumed to be due to a high sensitivity to Ni2+ ions. It is known that S. aureus showed a comparative weak resistance to antibiotics, and that E. coli was strong in chemical stress (Becerra et al., 2003). On the contrary, the antibacterial activity of Ni2+ ion toward E. coli was found to be stronger than it was toward S. aureus. In our study, the antibacterial activity of S. aureus was very similar to that of E. coli. From these results, it is convincing to believe that the antibacterial activity of Mg1−X NiX O solid solution is dependent on two factors: the generated amount of O2 − , and the amount of Ni2+ ions eluted from powder samples. In addition, the pH value of Mg1−X NiX O solid solution is also a predominant factor affecting antibacterial activity, because the stability of generated O2 − in aqueous solution was dependent on the pH value.
4.
Survival ratio, N/N0 / -
1
0.1
0
600
1200
1800
2400
3000
3600
Time / s Fig. 10 – Survival ratio of bacteria, using nickel acetate with a concentration of 50 × 10−5 mol dm−3 . ♦: S. aureus and : E. coli.
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Conclusions
Mg1−X NiX O solid solution powders at a molar ratio (MgO/NiO) ranging from 19 to 0.25 were prepared by heating at 1300 ◦ C for 12 h in air. By XRD measurement, a single phase of a cubic structure was detected in all powders. An increase in the doping amount of NiO resulted in a decrease of the lattice constant. The specific surface areas of all powders were nearly identical. The pH value was found to decrease when the doping amount of NiO in solid solution was increased. From ICP-AES measurement, the amount of Ni2+ ions eluted from the powders increased until it became 50% of the doping amount of NiO. In antibacterial tests, the antibacterial activity of Mg1−X NiX O solid solution decreased when the doping amount of NiO was increased, irrespective of the kind of bacteria. From the result of antibacterial tests using nickel acetate, antibacterial action toward E. coli on Ni2+ ions was stronger than it was toward S. aureus. Our study clarified that antibacterial activity of Mg1−X NiX O solid solution is dependent on two factors: the generated amount of O2 − , and the amount of Ni2+ ions eluted from Mg1−X NiX O solid solution. The reduction of antibacterial activity in our study corresponded to a decrease of pH values, which had a correlation with the generated amount of O2 − . That is, it is concluded that the pH value is also one of the predominant factors affecting the antibacterial activity of Mg1−X NiX O solid solution.
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References Atmaca, S., Gül, K., C¸ic¸ek, R., 1998. The effect of zinc on microbial growth. Turk. J. Med. Sci. 28, 595–598. Becerra, M.C., Eraso, A.J., Albesa, I., 2003. Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity. Luminescence 18, 334–340. Chang, C.W., Chang, W.L., Chang, S.T., 2008. Influence of pH on bioactivity of cinnamon oil against Legionella pneumophila and its disinfection efficacy in hot springs. Water Res. 2, 5022–5030. Chandra, S., Raizada, S., Tyagi, M., Sharma, P.K., 2008. Spectroscopic and biological approach of Ni(II) and Cu(II) complexes of 2-pyridinecarboxaldehyde thiosemicarbazone. Spectrochim. Acta A 69, 816–821. Hu, Y.H., Ruckenstein, E., 1996. Temperature-programmed desorption of CO absorbed on NiO/MgO. J. Catal. 163, 306–311. Katyal, N.K., Ahluwalia, S.C., Parkash, R., 1998. Solid solution and hydration behavior of magnesium-bearing tricalcium silicate phase. Cem. Concr. Res. 28, 867–875. Lee Yee-lean Cesario, T., Owens, M.D., Shanbrom, J., Thrupp, E.L.D., 2002. Antibacterial activity of citrate and acetate. Nutrition 18, 665–666. Liu, T.S., Stokes, R.J., Li, C.H., 1964. Fabrication and plastic behavior of single-crystal MgO–NiO and MgO–MnO solid-solution alloys. J. Am. Ceram. Soc. 47, 276–279. Mazzola, P.G., Jozala, A.F., de, Novaes L.C.L., Moriel, P., Penna, T.C.V., 2009. Minimal inhibitory concentration (MIC) determination of disinfectant and/or sterilizing agents. Braz. J. Pharm. Sci. 45, 241–248. Nurunnabi, M., Li, B., Kunimori, K., Suzuki, K., Fujimoto, K., Tomishige, K., 2005. Performance of NiO–MgO solid solution-supported Pt catalysts in oxidative steam reforming of methane. Appl. Catal. A: Gen. 292, 272–280. Ohira, T., Kawamura, M., Iida, Y., Fukuda, M., Yamamoto, O., 2008. Influence of the mixing ratio on antibacterial characteristics of MgO–ZnO solid solution in two phase coexistence region. J. Ceram. Soc. Jpn. 116, 1234–1237. Rahn, O., 1929. The size of bacteria as the cause of the logarithmic order of death. J. Gen. Physiol. 20, 179–205. Rao, K.V., Sunandana, C.S., 2008. XRD, microstructural and EPR susceptibility characterization of combustion synthesized nanoscale Mg1−X NiX O solid solutions. J. Phys. Chem. Solids 69, 87–96. Riley, D.P., Rivers, W.J., Weiss, R.H., 1991. Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems. Anal. Biochem. 196, 344–349.
Roe, A.J., O’Byrne, C., Mclaggan, D., Booth, I.R., 2002. Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity. Microbiology 148, 2215–2222. Saene, J., Veringa, S., Saene, H., Verhoef, J., Lerk, C., 1985. Effect of chlorhexidine and acetic acid on phagocytosis by polymorphonuclear leucocytes. Eur. J. Clin. Microbiol. Infect. Diseases 4, 493–497. Sawai, J., Igarashi, H., Hashimoto, A., Kokugan, T., Shimizu, M., 1996a. Effect of particle size and heating temperature of ceramic powders on antibacterial activity of their slurries. J. Chem. Eng. Jpn. 29, 251–256. Sawai, J., Kawada, E., Kanou, F., Igarashi, H., Hashimoto, A., Kokugan, T., Shimizu, M., 1996b. Detection of active oxygen generated from ceramic powders having antibacterial activity. J. Chem. Eng. Jpn. 29, 627–633. Sawai, J., Kojima, H., Ishizu, N., Itoh, M., Igarashi, H., Sawaki, T., Shimizu, M., 1997a. Bactericidal action of magnesium oxide powder. J. Inorg. Biochem. 71, 443. Sawai, J., Shiga, H., Kojima, H., 1997b. Escherichia coli damage by ceramic powder slurries. J. Chem. Eng. Jpn. 30, 1034–1039. Sawai, J., Shiga, H., Kojima, H., 2001. Kinetic analysis of death of bacteria in CaO powders slurry. Int. Biodeterior. Biodegradation 47, 23–26. Sawai, J., Himizu, K., Yamamoto, O., 2005. Kinetics of bacterial death by heated dolomite powder slurry. Soil Biol. Biochem. 37, 1484–1489. Seven, O., Dindar, B., Aydemir, S., Metin, D., Ozinel, M.A., Icli, S., 2004. Solar photocatalytic disinfection of a group of bacteria and fungi aqueous suspensions with TiO2 , ZnO and Sahara desert dust. J. Photochem. Photobiol. A: Chem. 165, 103–107. Small, P., Blankenhorn, D., Welty, D., Zinser, E., Slonczewski, J.L., 1994. Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. J. Bacteriol. 176, 1729–1737. Sönmez, M., Berber, I˙., Akbas¸, E., 2006. Synthesis, antibacterial and antifungal activity of some new pyridazinone metal complexes. Eur. J. Med. Chem. 41, 101–105. Tamai, H., Katsu, N., Ono, K., Yasuda, H., 2001. Antibacterial activated carbons prepared from pitch containing organometallics. Carbon 39, 1963–1969. Yamamoto, O., Hotta, M., Sawai, J., Sasamoto, T., Kojima, H., 1998. Influence of powder characteristic of ZnO on antibacterial activity. J. Ceram. Soc. Jpn. 106, 1007–1011 (in Japanese).
Contents lists available at SciVerse ScienceDirect
Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd
Effective factor on antibacterial characteristics of Mg1−X NiX O solid solution Toshiaki Ohira a,∗ , Osamu Yamamoto b a
Center for Geo-Environmental Science, Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata Gakuen-machi, Akita 010-8502, Japan b Department of Bio-System Engineering, Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa-shi, Yamagata 992-8510, Japan
a b s t r a c t Mg1−X NiX O solid solution powder samples with different chemical compositions were prepared by heating MgO–NiO mixtures at 1300 ◦ C for 12 h in air. From XRD measurement, all powder samples were indexed as a single phase of cubic structure, of which the diffraction peaks shifted to high-angle side with the increase of doping amount of NiO. The pH values of the solution dispersed with the powder samples decreased when the doping amount of NiO in solid solution was increased. Antibacterial activity of the powder samples was examined by colony count method. In the result, the antibacterial activity of Mg1−X NiX O was remarkably weaker than original MgO powders, irrespective of the kind of bacteria. In addition, it was found that the antibacterial activity of Mg1−X NiX O reduced with increasing the doping amount of NiO. Two factors, the generated amount of O2 − and the eluted amount of Ni2+ ions affected the antibacterial activity of Mg1−X NiX O solid solution. Especially, the stability of O2 − in aqueous solution is dependent on pH value. Therefore, the strength of antibacterial activity was associated with the pH values in the dispersed solution of Mg1−X NiX O. © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: MgO; NiO; Solid solution; Antibacterial activity; Colony count
1.
Introduction
Recently, magnesium oxide (MgO) has attracted strong interest as an antibacterial ceramic, because it has shown antibacterial activity in only small amounts of powder without the presence of light (Sawai et al., 1997a; Tamai et al., 2001). Sawai et al. (1997b) reported that main factors contributing to the occurrence of antibacterial activity were assumed to be the increase of the pH value because of the hydration of MgO, and super-oxide (O2 − ) generated from the surface of MgO. And they found that such powder characteristics of MgO as particle size and powder concentration, affected the antibacterial activity toward Escherichia coli and Staphylococcus aureus (Sawai et al., 1996a). Furthermore, extensive research on the antibacterial activity of MgO has shown that cubictype solid solution in the MgO–ZnO system exhibited stronger antibacterial activity, when compared with the original MgO (Ohira et al., 2008). In detail, antibacterial activity of cubic-type
∗
Mg1−X ZnX O solid solution that is enhanced with an increased doping amount of ZnO, is probably caused by the increase in the generated amount of O2 − . Therefore, it is important to understand the effective factors of the solid solutions with MgO as one of the end members on antibacterial activity, in order to know the antibacterial mechanism of the Mg1−X MX O (with M = Zn, Ni, etc.) solid solutions. Except for this understanding of Mg1−X ZnX O solid solution, however, there are no studies on the antibacterial activity of other Mg1−X MX O solid solutions having a cubic structure. According to a phase diagram of the MgO–NiO system, MgO and NiO can form complete solid solution having a cubic structure (Liu et al., 1964). Since antibacterial activity of Mg1−X NiX O solid solution may be dependent on the doping amount of NiO, a solid solution with different chemical compositions was prepared by heating the mixtures of MgO and NiO at 1300 ◦ C for 12 h. Using the colony count method, the antibacterial activity of Mg1−X NiX O solid solution having different chemical
Corresponding author. Tel.: +81 18 889 2450; fax: +81 18 837 0409. E-mail address: [email protected] (T. Ohira). Received 9 August 2012; Received in revised form 19 October 2012; Accepted 25 November 2012 0263-8762/$ – see front matter © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cherd.2012.11.015
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Table 1 – Sample code, molar ratio and chemical composition of powder samples used in this study. Sample code
Molar ratio (MgO/NiO)
MN05 MN10 MN20 MN50 MN80
19.0 9.00 4.00 1.00 0.25
Chemical composition Mg0.95 Ni0.05 O Mg0.90 Ni0.10 O Mg0.80 Ni0.20 O Mg0.50 Ni0.50 O Mg0.20 Ni0.80 O
compositions was evaluated, with an emphasis on the effect of the chemical composition on the solid solution.
concentration of 0.63 g dm−3 were independently collected by filtration, and dried at 100 ◦ C for 12 h. The crystal phases of the dried powders were evaluated by XRD, in order to confirm the formation phase of Mg1−X NiX O after hydration, because Mg(OH)2 and Ni(OH)2 may be formed by the hydration of MgO and NiO, and this can be detected by XRD. Thus, the degree of hydration of the powder samples was evaluated. Inductively coupled plasma atomic emission spectrometry measurement (ICP-AES; Seiko Instruments, Inc., SPS 7700) was performed to measure the amount of Ni2+ ions eluted from the solid solution. This was done because the Ni2+ ions are known to show antibacterial activity (Sönmez et al., 2006).
2.
Materials and methods
2.3.
2.1.
Preparation of materials
Escherichia coli 745 (hereafter, E. coli), a Gram-negative bacterium, and Staphylococcus aureus 9779 (hereafter, S. aureus), a Gram-positive bacterium, were used in antibacterial tests. These test bacteria were cultured aerobically in LB medium containing 0.5% yeast extract (Becton, Dickinson and Co.), 1% bactopeptone (Becton, Dickinson and Co.), and 1% sodium chloride (NaCl; Wako Pure Chemical Ind., Ltd., purity: 99.9%) at 36 ◦ C for 48 h. Bacteria suspended in the medium were cultured at 36 ◦ C for 48 h on a reciprocal shaker, and rinsed four times with sterile water. Finally, the bacterial suspension was diluted with sterile water at a concentration of 107 cfu dm−3 (cfu: colony forming unit).
The sample code, molar ratio (MgO/NiO) and chemical composition of Mg1−X NiX O solid solution are summarized in Table 1. The number in the sample code represents the molar fraction of NiO. For example, MN20 represents the 80 mol%MgO–20 mol%NiO powder sample. The powder samples of Mg1−X NiX O solid solution were prepared using a solid-state reaction. The mixtures of MgO (Ube Chemical Ind, Ltd., purity: 99.9%) and NiO (Nacalai Tesque, Inc., purity: 97.0%) were heated at 1300 ◦ C for 12 h in air. As reference materials against the as-prepared powder samples, pure MgO and NiO powders were prepared under identical heat-treatment conditions. In this work, MgO–NiO mixtures were heated at 1300 ◦ C, which is below both the melting and boiling points for NiO and MgO. Stoichiometrically, therefore, chemical compositions based on Mg1−X NiX O (X = 0.05, 0.10, 0.20, 0.50 and 0.80), should be unchanged with the mixed ratio of MgO–NiO mixtures.
2.2.
Powder characterization
The formation phase of the powder samples was identified by X-ray diffraction measurement (XRD; Rigaku, RAD-C SYSTEM). The scanning rate was 1◦ /min from 2 = 20◦ to 80◦ and the operation voltage and current of XRD were 40 kV and 20 mA, respectively. The value of the lattice constant was calculated using the diffraction peaks detected on XRD. The lattice constants (a0 ) of MgO, NiO and five Mg1−X NiX O powders were calculated by Eq. (1): a0 = {(h2 + k2 + l2 ) × d2 }
1/2
(1)
where h, k, and l are Miller indices and d is the interplane distance in nm. The specific surface area of the powder samples was determined by measuring the adsorption isotherms of N2 at −196 ◦ C (BET; Beljapan, Inc., BELSORP-mini), because of one of the powder characteristics affecting antibacterial activity (Yamamoto et al., 1998). Since the pH value in a bacterial growth environment is known to affect antibacterial activity (Small et al., 1994), it is essential to measure the pH value of the solution dispersed with the powder samples. Powder samples were dispersed into distilled water at a concentration of 0.63 and 40 g dm−3 to make powder slurries. After keeping the dispersed solution for 1 h at 36 ◦ C, the pH value of the solution was measured. After pH measurement, the powder slurries with a
2.4.
Preparation of bacterial suspension
Antibacterial test
The bacterial suspension was added into sterile water containing powder samples at a concentration of 0.63–40 g dm−3 . Thus, the antibacterial test was started, and each suspension was kept at 36 ◦ C for different amounts of time (15, 30, 45 and 60 min) on a reciprocal shaker. A 10−4 dm3 suspension pipetted at specified times (15, 30, 45 and 60 min) was spread on a nutrient agar (NA, Eiken Chemical, Co.) against E. coli, and spread on a pearl-core plate count agar (PPCA, Eiken Chemical, Co.) against S. aureus. These agars were incubated at 36 ◦ C for 24 h without the presence of light. After incubation, the colonies formed on the agar were counted. In vitro antibacterial activity of the powder samples was assessed by calculating the survival ratio = N/N0 , where N and N0 are, respectively, the viable bacterial counts N (cfu dm−3 ) at a specified time, and the initial counts of bacteria N0 (cfu dm−3 ) (Seven et al., 2004).
2.5.
Antibacterial activity of Ni2+ ions
The prepared bacterial suspension (Section 2.3) was added into nickel acetate solution with a concentration of 50 × 10−5 mol dm−3 , and an antibacterial test using nickel acetate solution was started. After 15, 30, 45 and 60 min, a 10−4 dm3 solution was adopted, and spread on either a nutrient agar against E. coli, or on a pearl-core plate count agar against S. aureus. After incubating these agars at 36 ◦ C for 24 h without the presence of light, the colonies formed on the agar were counted. By means of the calculation described in Section 2.4, the survival ratio was estimated. Based on the degree of the decline of the survival ratio on E. coli and S. aureus, the sensitivity to Ni2+ ions was compared between E. coli and S. aureus.
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Intensity/Arb.Unit
(g) (f) (e) (d) (c)
8 7 6
5 4 3 2 1 0
(222)
(311)
(220)
(111)
(200)
(b)
(a) 20
30
40
50
60
70
Fig. 1 – XRD patterns of powder samples obtained by heating at 1300 ◦ C for 12 h in air: (a) MgO, (b) MN05, (c) MN10, (d) MN20, (e) MN50, (f) MN80 and (g) NiO.
3.
Results and discussion
3.1.
Powder characterization
Fig. 1 shows XRD patterns of the powder samples obtained by heating at 1300 ◦ C, together with the pure powders NiO and MgO, both obtained by identical heat-treatment. A single phase of a cubic structure was formed in all obtained powder samples. The diffraction peaks corresponding to MgO were very similar to those of NiO, and shifted to the high-angle side when the doping amount of NiO was increased. The value of the lattice constant on the powder samples was calculated using the 200 diffraction peaks, which were consistent with the values estimated from Vegard’s rule. As shown in Fig. 2, the lattice constant was found to decrease linearly with the increase of the doping amount of NiO. This is due to the fact that the ionic radius of the Mg2+ ion is larger than that of the Ni2+ ion. Indeed, the ionic radii of the Mg2+ and Ni2+ ions were 72 pm and 69 pm, respectively. Thus, the decrease in the lattice constant seems to be attributed to the substitution of Mg2+ 0.4215 0.4210 0.4205 0.4200 0.4195 0.4190 0.4185 0.4180 0.4175 0.00
0.20
0.40
0.60
0.80
1.00
x in Mg1-XNiXO Fig. 2 – Change in lattice constant of MgO.
0
0.5
1
Relative pressure, p/p0 / Fig. 3 – N2 adsorption isotherms at −196 ◦ C on powder samples. 䊉: MgO, : MN05, : MN10, : MN20 and ♦: MN50.
80
Diffraction Angle, 2θθ / ° (CuKα α )
Lattice constant / nm
9 Volume absorbed / cm3 g-1
(222)
(311)
(220)
(200)
(111)
10
:MgO(cubic) :NiO(cubic)
1.20
ions by Ni2+ ions. These results indicate that Mg1−X NiX O solid solutions (MN05, MN10, MN20, MN50 and MN80) were successfully formed, which are consistent with various reports on the Mg1−X NiX O solid solution (Hu and Ruckenstein, 1996; Nurunnabi et al., 2005; Rao and Sunandana, 2008). NiO is known to start melting at 1980 ◦ C at 1 atm. Thus, any heattreatment above 1980 ◦ C may be able to change the expected compositions based on Mg1−X NiX O stoichiometrically. Since the heating temperature performed in this study was 1300 ◦ C, the MgO/NiO ratio in the obtained Mg1−X NiX O solid solutions should be equal to the ratio in the MgO–NiO mixtures. Yamamoto et al. (1998) have reported antibacterial activity of ZnO enhanced with an increase in the specific surface area; this, because of the increase in the generated amount of H2 O2 on its surface. That is, the specific surface area of the powder samples is one of the powder characteristics affecting antibacterial activity. Therefore, measuring the specific surface area of powder samples is important for precisely evaluating antibacterial activity. By BET measurement, the specific surface area of the obtained five solid solutions was below 1 m2 g−1 , having no accuracy for quantitative analysis. Fig. 3 shows the obtained N2 adsorption isotherms at −196 ◦ C of pure MgO, MN05, MN10, MN20 and MN50. It was found that the amount of N2 adsorbed on MgO was much larger than that on Mg1−X NiX O. On the other hand, there was little difference in the amount of the adsorbed N2 in Mg1−X NiX O. From these results, the specific surface areas of Mg1−X NiX O powder samples were judged to be very similar. It has been reported that increasing the pH value causes the enhancement of antibacterial activity (Sawai et al., 2001). Thus, measuring the pH value in aqueous solution containing the powder samples is essential to evaluate antibacterial activity. As summarized in Table 2, the pH values of the powder samples decreased when the doping amount of NiO in solid solution was increased. Fig. 4 shows XRD patterns of the dried powders after pH measurement. In the case of the original MgO, diffraction peaks corresponding to Mg(OH)2 of a hexagonal type were detected. On the other hand, no peaks of Ni(OH)2 were detected in any of the powder samples. These results indicate that the degree of hydration in the original MgO was the highest for all powder samples. Therefore, the reason that original MgO showed the highest pH value might be assumed to be due to the formation of Mg(OH)2 resulting from the hydration
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Table 2 – Sample code, specific surface areas, pH values and eluted Ni2+ ion concentration of powder samples in this study. Specific surface area (m2 g−1 )
Sample code
0.63 g dm−3 MgO MN05 MN10 MN20 MN50 MN80 NiO
8.4 <1 <1 <1 <1 <1 <1
11 8.8 8.0 7.5 6.2 5.5 5.5
Intensity/Arb.Unit
of MgO. On the other hand, there were no diffraction peaks in either Mg(OH)2 or Ni(OH)2 in MN05, MN10, MN20, MN50 and MN80. This may mean that the formation of Mg1−X NiX O solid solution inhibits hydration. Therefore, the inhibition of hydration might be related to the decrease of pH values in the Mg1−X NiX O powder samples. This implies that the effect of pH values on the antibacterial activity became weak as the doping amount of NiO was increased. Chandra et al. (2008) have suggested that Ni2+ ions show only slight antibacterial activity. Therefore, the amount of Ni2+ ions eluted from the powder samples was measured by ICP-AES. The amount of Ni2+ ions eluted from the powder samples increased, reaching 50% of the doping amount of NiO. It was also found that the amount of eluted Ni2+ ions decreased. Regarding NiO itself, the amount of eluted Ni2+ ions was the smallest of all the powder samples. It has been reported that the MgO content in solid solution affects the degree of hydration (Katyal et al., 1998). In our comparison of pH values between the powder concentrations of 0.63 and 40 g dm−3 , similar values were observed in both MN80 and NiO. That is, pH values of dispersions with the powder concentration of 0.63 and 40 g dm−3 were 5.5 and 5.7, respectively.
20
40
60
80
Diffraction Angle, 2θθ / ° (CuKα α ) Fig. 4 – XRD patterns of powder samples after dispersed into distilled water at a concentration of 0.63 g dm−3 .
Eluted Ni2+ ion concentration (mol dm−3 )
pH value 40 g dm−3 – 9.7 8.7 8.4 6.8 5.7 5.7
– 5.1 × 10−5 12 × 10−5 13 × 10−5 18 × 10−5 8.7 × 10−5 3.3 × 10−5
On the other hand, the pH values of MN05, MN10, MN20 and MN50 significantly increased when the powder concentration was increased. This result seems to indicate a higher hydration resistance for MN80 and NiO. If the hydration resistance of Mg1−X NiX O solid solution is high, the solubility should be low. Therefore, the reason that the eluted amount of Ni2+ ions decreased in MN80 and NiO might be presumed to be due to their high hydration resistance.
3.2.
Antibacterial activity
The relative strength of antibacterial activity on Mg1−X NiX O solid solution having different chemical compositions was evaluated by the colony count method. When the survival ratio changes with a steep decrease at a specified time, the colony count method helps us see strong antibacterial activity in powder samples. Fig. 5(a) and (b) shows the survival ratio of S. aureus and E. coli, respectively, using pure MgO and NiO powders suspended at the powder concentration of 0.63 g dm−3 . In both S. aureus and E. coli, the reduction of the survival ratio on MgO was much larger than that on NiO. That is, MgO showed antibacterial activity, while no antibacterial activity was recognized in NiO. Fig. 6(a) and (b) shows the survival ratios of S. aureus and E. coli, respectively, using five solid solutions suspended at the powder concentration of 0.63 g dm−3 . In S. aureus, the values of the survival ratio on MN05, MN10 and MN20 decreased over time. There was no significant decrease observed in either MN50 or MN80. In a comparison of the survival ratios at specified times for MN05, MN10 and MN20, the survival ratio was found to decrease when the doping amount of NiO in solid solution was increased, although the behavior resulting from the reduction of MN05 was similar to that of MN10. In the case of E. coli, the reduction behavior of the survival ratio was almost identical with that of S. aureus. That is, the antibacterial activity toward E. coli was found to be less with an increased amount of NiO doping. MN50 and MN80 did not show any significant antibacterial activity. Regarding the antibacterial activity of MgO, it is reported that the generation of O2 − , as an antibacterial agent, was detected on its surface (Sawai et al., 1996b). In the case of the Mg1−X NiX O solid solution, the O2 − generated from the surface of the solid solution, therefore, might be a chemical species contributing to the occurrence of antibacterial activity. Antibacterial activity should be enhanced with an increase of the specific surface area of powder samples because of the increase in the amount of active oxygen such as O2 − and H2 O2 generated on its surface. As described in Section 3.1, the specific surface area values of Mg1−X NiX O solid solutions were of similar. This implies that the effect of specific surface areas on antibacterial activity is probably also similar. However, antibacterial activity
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Fig. 5 – Survival ratio of (a) S. aureus and (b) E. coli on MgO and NiO powder samples slurries at powder concentration of 0.63 g dm−3 . : MgO and 䊉: NiO. of the five solid solutions was less when the doping amount of NiO was increased. These results indicate that there is an effective factor contributing to the occurrence of antibacterial activity besides the specific surface area. In the comparison of the survival ratios at specified times between pure MgO and the five solid solutions (see Figs. 5 and 6), the decrease of the survival ratio on the five solid solutions is smaller than that of pure MgO. This means that the antibacterial activity of Mg1−X NiX O solid solution was weaker than that of pure MgO. Chang et al. (2008) reported that the culturability of Legionella pneumophila decreased when the pH values increased in the range from 6 to 10. It has also been reported that the effect of pH values on antibacterial activity was remarkably enhanced when pH values were greater than 10 (Small et al., 1994; Sawai et al., 2001). The pH value of MgO was 11 in the present study, and showed the strongest antibacterial activity of all powder samples. When increasing the doping amount of NiO, the pH values of the five solid solutions decreased. Similarly, the antibacterial activity on the five solid solutions decreased when the doping amount of NiO was increased, although MN50 and MN80 did not show significant antibacterial activity. From these results, it is reasonable
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Fig. 6 – Survival ratio of (a) S. aureus and (b) E. coli on solid solution slurries at powder concentration of 0.63 g dm−3 . : MN05, : MN10, ♦: MN20, : MN50 and ×: MN80. to believe that a decrease of pH values is related to a reduction of antibacterial activity when the doping amount of NiO is increased. That is to say, the antibacterial activity of the powder samples might be dependent on the pH values. Figs. 7 and 8 show changes in the survival ratio of S. aureus and E. coli having different powder concentrations, using MN05 and MN20. The survival ratio was found to decrease when the powder concentration increased, irrespective of the kind of bacteria, indicating that the antibacterial activity of MN05 and MN20 was enhanced with an increase of powder concentration. As described in Table 2, the pH values of five solid solutions were below 10; this is known to be inadequate to exhibit antibacterial activity for S. aureus and E. coli (Small et al., 1994; Sawai et al., 2001). This result implies that the generated amount of chemical species that contributes to the occurrence of antibacterial activity increased with the increase of powder concentration. As described above, the chemical species contributing the occurrence of antibacterial activity in the Mg1−X NiX O solid solution should be O2 − generated from the surface of the solid solution. It has been reported that a self-dismutation reaction of O2 − proceeds rapidly when the pH value in an aqueous solution decreases from 10 to 7
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Fig. 7 – Survival ratio of S. aureus on (a) MN05 and (b) MN20 slurries at each powder concentration. : 0.63 g dm−3 , : 2.5 g dm−3 , :10 g dm−3 and 䊉: 40 g dm−3 . (Riley et al., 1991). As expressed by the following equation (2), the self-dismutation reaction of O2 − results in the formation of hydro peroxide (H2 O2 ). And H2 O2 is also known to show antibacterial activity toward E. coli and S. aureus (Mazzola et al., 2009). 2O2 − + 2H+ → H2 O2 + O2
(2)
Since the pH value of the dispersed solution of MN20 was lower than that of MN05, the self-dismutation reaction of O2 − on MN20 might be faster than that on MN05. In other words, O2 − in the dispersed solution of MN05 was more stable, when compared to that of MN20. Assuming that H2 O2 generated by the self-dismutation reaction of O2 − contributed to antibacterial activity, the antibacterial effect of H2 O2 might be less than that of O2 − , depending on the amount of O2 − . Thus, the reason for the enhancement of antibacterial activity with an increased powder concentration may be associated with the stability of O2 − , in addition to the increased generated amount of O2 − on the surface. Generally, the death of bacteria has been reported to be logarithmic in order (Rahn, 1929). When the survival ratios decrease linearly (Figs. 7 and 8), the death rate of bacteria can
Fig. 8 – Survival ratio of E. coli on (a) MN05 and (b) MN20 slurries at each powder concentration. : 0.63 g dm−3 , : 2.5 g dm−3 , :10 g dm−3 and 䊉: 40 g dm−3 . be understood by first-order kinetics as dN/dt = −kN: where t, N and k are, respectively, time, survival ratio, and the death rate constant (Sawai et al., 2005). Thus, a high k value means strong antibacterial activity. Fig. 9(a) and (b) shows the relationship between k values and powder concentrations toward S. aureus and E. coli, respectively. The k values of MN05 were always higher than those of MN20 in the range of powder concentration performed in this study, irrespective of the kind of bacteria; that is, the antibacterial action of MN05 was stronger than that of MN20. In Fig. 9(a), the slope showing S. aureus has the identical value of 0.65 for both MN05 and MN20. In the case of E. coli (see. Fig. 9(b)), the slope degree of MN20 is higher than that of MN05. The slope of the line indicates the sensitivity to chemical species that contribute to antibacterial activity. It is believed that the difference of the slope degree reflects changes in sensitivity to the eluted Ni2+ ions irrespective of the amount of generated O2 − , because the amount of Ni2+ ions eluted from MN20 was found to be greater than that eluted from MN05. Fig. 10 shows the survival ratios of S. aureus and E. coli when using nickel acetate at a concentration of 50 × 10−5 mol dm−3 . In comparing the survival ratios of S. aureus and E. coli, the ratio of S. aureus was less than that of E. coli; that is, the antibacterial
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Fig. 9 – Relationship between powder concentration and death rate constant (k) for (a) S. aureus and (b) E. coli. : MN05 and ; MN20.
action of the Ni2+ ion toward E. coli was stronger than it was toward S. aureus. Acetate salts such as zinc acetate and potassium acetate have been known as a chemical species during the occurrence of antibacterial activity (Atmaca et al., 1998; Lee Yee-lean Cesario et al., 2002; Roe et al., 2002). In these reports, an acetate concentration above 230 × 10−5 mol dm−3 for zinc acetate and 130,000 × 10−5 mol dm−3 for potassium acetate showed antibacterial activity toward S. aureus. In addition to these reports, it has been revealed that acetic acid with a concentration of 830 × 10−5 mol dm−3 has no bactericidal effect (Saene et al., 1985). Since these concentrations were remarkably higher than 50 × 10−5 mol dm−3 , the effect of the acetate ion on antibacterial activity can be ignored for those antibacterial tests in our study where nickel acetate was used. In Fig. 10, therefore, the occurrence of antibacterial activity was presumed to be due to Ni2+ ions. And the antibacterial action of the Ni2+ ion toward E. coli as Gram-negative bacteria, was stronger than it was toward S. aureus as Grampositive bacteria. Consequently, the reason that the slope degree when using E. coli on MN20 was higher than when used on MN05, may be assumed to be due to a high sensitivity to Ni2+ ions. It is known that S. aureus showed a comparative weak resistance to antibiotics, and that E. coli was strong in chemical stress (Becerra et al., 2003). On the contrary, the antibacterial activity of Ni2+ ion toward E. coli was found to be stronger than it was toward S. aureus. In our study, the antibacterial activity of S. aureus was very similar to that of E. coli. From these results, it is convincing to believe that the antibacterial activity of Mg1−X NiX O solid solution is dependent on two factors: the generated amount of O2 − , and the amount of Ni2+ ions eluted from powder samples. In addition, the pH value of Mg1−X NiX O solid solution is also a predominant factor affecting antibacterial activity, because the stability of generated O2 − in aqueous solution was dependent on the pH value.
4.
Survival ratio, N/N0 / -
1
0.1
0
600
1200
1800
2400
3000
3600
Time / s Fig. 10 – Survival ratio of bacteria, using nickel acetate with a concentration of 50 × 10−5 mol dm−3 . ♦: S. aureus and : E. coli.
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Conclusions
Mg1−X NiX O solid solution powders at a molar ratio (MgO/NiO) ranging from 19 to 0.25 were prepared by heating at 1300 ◦ C for 12 h in air. By XRD measurement, a single phase of a cubic structure was detected in all powders. An increase in the doping amount of NiO resulted in a decrease of the lattice constant. The specific surface areas of all powders were nearly identical. The pH value was found to decrease when the doping amount of NiO in solid solution was increased. From ICP-AES measurement, the amount of Ni2+ ions eluted from the powders increased until it became 50% of the doping amount of NiO. In antibacterial tests, the antibacterial activity of Mg1−X NiX O solid solution decreased when the doping amount of NiO was increased, irrespective of the kind of bacteria. From the result of antibacterial tests using nickel acetate, antibacterial action toward E. coli on Ni2+ ions was stronger than it was toward S. aureus. Our study clarified that antibacterial activity of Mg1−X NiX O solid solution is dependent on two factors: the generated amount of O2 − , and the amount of Ni2+ ions eluted from Mg1−X NiX O solid solution. The reduction of antibacterial activity in our study corresponded to a decrease of pH values, which had a correlation with the generated amount of O2 − . That is, it is concluded that the pH value is also one of the predominant factors affecting the antibacterial activity of Mg1−X NiX O solid solution.
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