Catalytic sterilization of Escherichia coli K 12 on Ag/Al₂O₃ surface

Abstract

Bactericidal action of Al₂O₃, Ag/Al₂O₃ and AgCl/Al₂O₃ on pure culture of Escherichia coli K 12 was studied. Ag/Al₂O₃ and AgCl/Al₂O₃ demonstrated a stronger bactericidal activity than Al₂O₃. The colony-forming ability of E. coli was completely lost in 0.5 min on both of Ag/Al₂O₃ and AgCl/Al₂O₃ at room temperature in air. The configuration of the bacteria on the catalyst surface was observed using scanning electron microscopy (SEM). Reactive oxygen species (ROS) play an important role in the expression of the bactericidal activity on the surface of catalysts by assay with O₂/N₂ bubbling and scavenger for ROS. Furthermore, the formation of CO₂ as an oxidation product could be detected by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and be deduced by total carbon analysis. These results strongly support that the bactericidal process on the surface of Ag/Al₂O₃ and AgCl/Al₂O₃ was caused by the catalytic oxidation.

1. Introduction

Sterilization is an important procedure to maintain a sanitary environment. For this purpose, many chemical germicides have been used. With these applications, the potential dangers of the compounds to human health and environment are concerned. TiO₂ photocatalysts applied in water disinfection process were noticeable, which provided an alternative to conventional chemical germicides [1], [2], [3], [4], [5]. However, this technology requires a relatively complex device and photo energy. Recently, we have reported the inactivation efficiency of Ag/Al₂O₃ and Cu/Al₂O₃ to SARS coronavirus, bacteria and yeast [6]. The microbes are completely inactivated in 5 min on Ag/Al₂O₃ surface at room temperature in air, and we suggest a catalytic oxidation mechanism in this disinfection process.

Even though the bactericidal effect of compounds containing silver has been investigated for a long time, its mechanism is still controversial. Some reports insisted that the antibacterial activity of silver-loaded compounds is depended on the cytotoxicity of Ag⁺ eluted from the compounds into system containing the microorganisms [7], [8]. At the same time, it has been suggested that the reactive oxygen species play an important role in the expression of the bactericidal activity in water [9], [10].

To identify the inactivation mechanism of bactericidal process on the surface of Ag/Al₂O₃ in air, e.g. by catalytic oxidation or by toxicity of heavy metals, Ag/Al₂O₃ and AgCl/Al₂O₃ were used in this study since they have different amount of Ag⁺ which may be eluted from the catalysts. In this study, we also report that reactive oxygen species (ROS) play an important role in the expression of the bactericidal activity on the surface of catalysts.

2. Experimental

2.1. Catalysts

Ag/Al₂O₃ and AgCl/Al₂O₃ (Ag 5 wt%) were prepared by impregnation and precipitation methods respectively. After Al₂O₃ powder was introduced into an appropriate amount of silver nitrate aqueous solution, the mixture was stirred at room temperature. As for AgCl/Al₂O₃, an appropriate amount of ammonium chloride aqueous solution was added into the mixture at a rate of 0.5 ml/min, while the Ag/Al₂O₃ need not this step. Both of the wet samples were dried at 120 °C for 12 h, and then calcined in air at 600 °C for 3 h [11]. Before using, Al₂O₃, Ag/Al₂O₃ and AgCl/Al₂O₃ powders were pressed into wafers of ca. 20 mg/cm². XRD is used to investigate the silver phase on γ-Al₂O₃. In the case of Ag/Al₂O₃, only the γ-Al₂O₃ phase was detected when the silver loading was 5 wt%, and the absence of diffraction lines of silver phase on 5 wt% Ag/Al₂O₃ catalyst indicates that silver is at a very high dispersion degree. To 5 wt% AgCl/Al₂O₃, however, the peaks attributable to AgCl phase was observed at 2θ of 27.76°, 32.24°, 55.86°, 57.34° and 76.54° (PDF-ICDD 85-1355, PDF-ICDD 31-1238). The absence of diffraction lines of silver phase on 5 wt% Ag/Al₂O₃ catalyst indicates that silver is at a very high dispersion degree. TEM was also used to investigate the silver phase on γ-Al₂O₃. As for 5 wt% Ag/Al₂O₃, the silver particle (ranging from 15 to 20 nm) at a very high dispersion degree was observed. In the case of 5 wt% AgCl/Al₂O₃, the silver particle was greater than that of 5 wt% Ag/Al₂O₃ (ranging from 20 to 50 nm).

2.2. Culture of micro-organisms

E. coli K 12 strain ATCC23716 was inoculated into LB broth (Fluka Co. 61748) and grew aerobically for 24 h at 37 °C with constant agitation. Aliquots of the culture were inoculated into fresh medium and incubated at 37 °C for 4–5 h until reaching an exponential growth phase. Bacterial cells were collected using centrifugation at 10,000 rpm for 10 min, and the bacterial pellet was washed with sterile water. Finally, bacterial cells were suspended in the water then diluted to the required cell density corresponding to 10⁸ colony forming units per milliliter (CFU/ml).

2.3. Bactericidal experiment and analysis method

A 20 μl aliquot of the E. coli suspension was applied onto the wafer surface of catalyst, and the contacting times (refers to the bacteria contacting time with the wafers of catalyst before sampling) were for 0.5, 2, 5, 10 and 20 min at room temperature. The survival cells were washed off from the catalyst wafers with sterile water. 0.5 ml eluate was immediately injected into 4.5 ml 0.9% NaCl aqueous solution to eliminate the effect of Ag⁺ [12], and then plated on LB agar (Fluka Co.61746) plates. The plates were incubated at 37 °C for 24 h before counting. All experiments were repeated three times. The wafers of catalysts were investigated using SEM during the bactericidal process. Before SEM measurement, the E. coli on the wafers were fixed with glutaraldehyde and osmium tetroxide, drained with ethanol/water with the concentrations of ethanol increasing. The absolute ethanol was replaced by dimethoxymethane, and the samples underwent critical point drying with CO₂. The wafers were glued onto stages with conductive silver and metallized with gold. The samples were microscoped and photographed with a scanning electron microscope (Fei QUANTA 200).

The bactericidal activity was measured under either aerobic or anaerobic conditions, which was achieved by oxygen or nitrogen bubbling to investigate the effect of dissolved oxygen. Superoxide dismutase (SOD, Sigma) was used to investigate the effect of ROS on bactericidal activity. Fifty unit per ml and 100 unit/ml of SOD solution was made, respectively. The solution was added to the E. coli K12 suspension and mixed sufficiently. A 20 μl of the mixture of E. coli suspension and SOD solution was applied onto the wafer surface of catalyst, and the sampling times were for 0.5, 2, 5, 10 and 20 min at room temperature.

In situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) method was used to investigate the dynamic changes of IR spectra on Ag/Al₂O₃ surface during the bactericidal process, and DRIFTS spectra were recorded on a Nexus 670 (Thermo Nicolet) FT-IR, equipped with an in situ diffuse reflection chamber and a high sensitivity MCT detector. After E. coli suspension and catalyst powder were mixed completely, the mixed sample was placed in a ceramic crucible in the in situ chamber. Total carbon (TC) content of mixture of E. coli and catalysts is determined using Apollo 9000 Analyzer (Terkmar Dohmann) during the bactericidal process. It was observed that TC decreased during the catalytic oxidation process, which implied that TC of E. coli had converted to CO₂ and released from the catalysts. The formation of CO₂ during this process can be calculated using the following equation:

$$\mathrm{formation}\mathrm{of}{\mathrm{CO}}_2/\%=\frac{{\mathrm{TC}}_0-{\mathrm{TC}}_i}{{\mathrm{TC}}_0}\times 100\%\text{,}$$

where TC₀ signifies initial TC and TC_i is TC at different times during this process.

3. Results and discussion

3.1. Bactericidal effects of the catalysts

The bactericidal activity of Al₂O₃, Ag/Al₂O₃ and AgCl/Al₂O₃ in air was investigated. Fig. 1 shows the time course changes with the viable cell count of E. coli. The process where Al₂O₃ used as host compounds (Fig. 1a) shows that the cell concentration decreased from 2.8 × 10⁸ CFU/ml to 1 × 10⁶ CFU/ml within 20 min after addition. Obviously, the processes with Ag/Al₂O₃ (Fig. 1b) and AgCl/Al₂O₃ (Fig. 1c) show a higher bactericidal effect on E. coli than Al₂O₃. The bacteria lost their colony forming ability in 0.5 min after applied onto the wafer of Ag/Al₂O₃ and AgCl/Al₂O_3. The spoiling of bacteria was also observed by SEM photographs as shown in Fig. 2 .

Fig. 1.

E. coli K 12 inactivation on (a) Al₂O₃, (b) AgCl/Al₂O₃ and (c) Ag/Al₂O₃ surface. Initial concentration of bacteria: 2.8 × 10⁸ CFU/ml.

Fig. 2.

SEM images of E. coli adsorbed on (a) Al₂O₃ at room temperature for 0.5 min and (b) 24 h, on (c) AgCl/Al₂O₃ for 0.5 min, (d) for 0.5 h, (e) for 1 h and on (f) Ag/Al₂O₃ for 0.5 min, (g) for 0.5 h and (h) for 1 h.

Fig. 2a and b show the SEM images of E. coli adhered on Al₂O₃ at room temperature for 0.5 min and 24 h, respectively. Although some cells were damaged after E. coli exposured to the wafer of Al₂O₃ for 0.5 min, the viable cells were still detectable using plate counting method. Some cells collapsed and led to release of intracellular constituents, however, their membrane and intracellular constituents covered the surface even after 24 h. Fig. 2c–e and f–h show the SEM images of E. coli adhered on AgCl/Al₂O₃ and Ag/Al₂O₃ at room temperature for 0.5 min, 0.5 and 1 h, respectively. As shown in Fig. 1, it is obvious that Ag/Al₂O₃ and AgCl/Al₂O₃ gave a higher bactericidal effect on E. coli than Al₂O₃. Although AgCl/Al₂O₃ exhibited a similar bactericidal ability comparing with Ag/Al₂O₃, the SEM images indicate that different mechanisms are involved in the bactericidal action of AgCl/Al₂O₃ and Ag/Al₂O₃. The cell membrane damage mainly started at one end of E. coli on the surface of AgCl/Al₂O₃ (Fig. 2c), and intracellular constituents were released from the damaged site. After exposure time of 0.5 h, the cell was further spoiled (Fig. 2d). After 1 h, the clean catalyst surface reappeared (Fig. 2e). The damage of bacteria on the surface of Ag/Al₂O₃ was more serious than that of AgCl/Al₂O₃ (Fig. 2f–h). Even though after 30 s on AgCl/Al₂O₃, a great deal of intracellular constituents spouted from damaged sites on the cell membrane (Fig. 2f). After exposure time of 0.5 h, E. coli cells shrank and the catalyst surface covered by the cells reappeared (Fig. 2g, h).

As mentioned above, the bactericidal mechanism of Ag containing compounds is still controversial [8], [9], [10], [11]. During the process of the E. coli suspension contacted with Ag/Al₂O₃ and AgCl/Al₂O₃, Ag⁺ released unavoidablely. In order to confirm the bactericidal effect of Ag⁺, the catalyst wafers were dip in sterile water for 10 min and the quantitative analysis of Ag⁺ in the eluate was carried out with an atomic adsorption spectrophotometer. The bactericidal effect of a series of AgNO₃ (10⁻⁹, 10⁻⁸, 10⁻⁵, 10⁻⁴, 10⁻³  M) contacting with E. coli suspension for 10 min were determined. The correlation between the concentration of Ag⁺ and the quantity of survival cells was reflected in a curve, so the effect of Ag⁺ released from the catalyst can be confirmed. As shown in Fig. 3 , the colony-forming ability of E. coli lost in Ag⁺ containing solution when the concentration of Ag⁺ is more than 10⁻⁴  M. The concentrations of Ag⁺ eluted from Ag/Al₂O₃ and AgCl/Al₂O₃ were less than 2.9 × 10⁻⁴  M and 4.7 × 10⁻⁷  M under such experimental conditions, respectively. According to this result, Ag⁺ was released from the catalysts, and the bactericidal ability of the Ag/Al₂O₃ would be related to the amount of Ag⁺ dissolved in a certain extent. However the amount of Ag⁺ eluted from AgCl/Al₂O₃ is too little to cause any bactericidal effect against E. coli.

Fig. 3.

Effect of the concentration of Ag⁺ on the colony-forming ability of E. coli.

3.2. Effect of oxygen and nitrogen gas bubbling

The effect of oxygen and nitrogen gas bubbling on the bactericidal activity was also investigated. This investigation was performed to obtain information about the role of oxygen in the bactericidal activity of Ag/Al₂O₃ and AgCl/Al₂O₃. 0.5 g Al₂O₃, Ag/Al₂O₃ and AgCl/Al₂O₃ powder was added into 40 ml sterilized water respectively, and then the aerobic and anaerobic condition was achieved by oxygen and nitrogen gas bubbling for 40 min before piping the bacteria into the system. The bubbling was continued throughout the treatment period. The bactericidal activity of Al₂O₃ under the same condition was studied as the control experiment. The results are shown in Fig. 4 . Under the anaerobic condition, the bactericidal activity of Ag/Al₂O₃ sharply decreased compared to that in the aerobic condition. Though the bactericidal activity of AgCl/Al₂O₃ was lower than Ag/Al₂O₃, it can also be obviously observed that the bactericidal activity in aerobic condition is better than that in anaerobic condition. These results suggest that nitrogen gas bubbling drastically suppresses the bactericidal activity of Ag/Al₂O₃, and indicate that the presence of oxygen is necessary for the bactericidal activity by Ag/Al₂O₃ and AgCl/Al₂O₃.

Fig. 4.

Effect of O₂ and N₂ bubbling on the bactericidal activity of Ag/Al₂O₃ and AgCl/Al₂O₃: (1) Al₂O₃, (2) AgCl/Al₂O₃, (3) Ag/Al₂O₃ in aerated condition; (4) Al₂O₃, (5) AgCl/Al₂O₃, (6) Ag/Al₂O₃ in nonaerated condition.

3.3. Effect of ROS on the surface of catalysts in the bactericidal process

From above experimental results, we consider that oxygen can be activated to ROS by the catalyst, which play an important role in the bactericidal activity. The ROS might contain superoxide anions, hydrogen peroxide and hydroxyl radicals. SOD is a scavenger for superoxide anions and hydroxyl radicals. The effect of addition of SOD on the bactericidal activity was investigated (Fig. 5 ). Compared with the situation without SOD, in the presence of SOD, the bacterial survival apparently increased on the surface of Ag/Al₂O₃ and AgCl/Al₂O₃, respectively. The addition of 100 unit/ml SOD drastically suppressed the bactericidal activity. The survival bacterial was kept more than 1 × 10⁶ CFU/ml when 100 unit/ml of SOD added (The survival bacteria were less than 5 × 10⁵ CFU/ml when 50 unit/ml of SOD added). This result indicates that the formation of superoxide anions and hydroxyl radicals contributes to the bactericidal activity.

Fig. 5.

Effect of SOD against ROS on the bactericidal activity of (a) Ag/Al₂O₃ and (b) AgCl/Al₂O₃, respectively. 0 unit/ml of SOD (The bactericidal survival in the case of 0 unit/ml of SOD reached 0.0E + 00 CFU/ml); 50 unit/ml of SOD; 100 unit/ml of SOD.

3.4. Formation of CO₂ during bactericidal process

As shown in Fig. 6 , Ag/Al₂O₃ added with E. coli was investigated using DRIFTS during exposure of the sample to O₂ for 42 h at 44 °C. After exposure of the sample to pure O₂ for 18 h, peaks at 2339 and 2362 cm⁻¹ assigned to gas phase CO₂ were observed, and the intensity of CO₂ peaks increased with time increasing. The appearance of gas phase CO₂ strongly supports that the bactericidal process on Ag/Al₂O₃ not only leads to the cell death, but also causes the completely catalytic oxidation of bacteria and other intermediates.

Fig. 6.

Dynamic changes of in situ DRIFTS spectra of Ag/Al₂O₃ as a function of time during exposure of the mixed sample to pure O₂ at 44 °C. Before measurement, the catalyst powder was mixed with E. coli suspension.

Furthermore, the formation of CO₂ was investigated by TC analysis as shown in Fig. 7 . About 49% and 28% of TC of E. coli were converted to CO₂ in air at 44 °C after the E. coli cells were added to the surfaces of Ag/Al₂O₃ and AgCl/Al₂O₃ for 125 h, respectively.

Fig. 7.

Detection of CO₂ produced during the bactericidal process at 44 °C by TC analysis: (♦) Ag/Al₂O₃; (■) AgCl/Al₂O₃; (▴) Al₂O₃.

It should be noticed that the time of surface reappearance is different from the time producing CO₂. In the SEM images, we can only see the surface uncovered with bacteria reappeared, but the surface might not as clean as the fresh surface, where small molecules might adsorb. The small molecules might have not been oxygenated completely into CO₂, but they already could not be seen using SEM. In addition, the catalyst could adsorb a mass of CO₂, because of its large specific surface area. Therefore the detection of CO₂ desorbed from the catalyst will be delayed.

4. Conclusion

Ag/Al₂O₃ and AgCl/Al₂O₃ demonstrated a stronger bactericidal activity than Al₂O₃. The colony-forming ability of E. coli was completely lost in 0.5 min on both of Ag/Al₂O₃ and AgCl/Al₂O₃ at room temperature in air. Relying on SEM investigation, the different bactericidal mechanisms were identified on the surfaces of Ag/Al₂O₃ and AgCl/Al₂O₃. The role of Ag⁺ eluted from AgCl/Al₂O₃ can be ignored in our experiments. Oxygen, which can be activated to ROS by catalyst, is essential for the expression of bactericidal activity. The strong catalytic oxidation ability was expressed on the Ag/Al₂O₃ surface, and CO₂ as the ultimate product was observed during the bactericidal process. All of these results indicate that the catalytic oxidation is the essential mechanism in bactericidal process. Further work is needed to clarify the details of these actions.

5. Abbreviations

ROS

reactive oxygen species

SEM

scanning electron microscopy

SARS

severe acute respiratory syndrome

XRD

X-ray polycrystalline diffraction

TEM

transmission electron microscopy

CFU/ml

colony forming units per milliliter

SOD

superoxide dismutase

DRIFTS

diffuse reflectance infrared Fourier transform spectroscopy

TC

total carbon