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Influence of reactive oxygen species on the sterilization of microbes
Current Applied Physics 13 (2013) S30eS35
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Influence of reactive oxygen species on the sterilization of microbes Han S. Uhm a, *, Eun H. Choi a, Guang S. Cho a, Daniel H. Hwang b a
Department of Electrophysics, Kwangwoon University, 447-1 Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of Korea Department of Nutrition and Western Human Nutrition Research Center, USDA ARS, University of California, One Shields Avenue, Davis, CA 95616-5270, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2012 Received in revised form 12 December 2012 Accepted 29 December 2012 Available online 7 January 2013
The influence of reactive oxygen species on living cells, including various microbes, is discussed. A sterilization experiment with bacterial endospores reveals that an argoneoxygen plasma jet very effectively kills endospores of Bacillus atrophaeus (ATCC 9372), thereby indicating that oxygen radicals are the key element of sterilization. Ozone in acidic water also kills endospores of B. atrophaeus very effectively, demonstrating the capability of cleaning a large surface area contaminated by toxic biological agents. The viable microbe numbers after the contact with acidic ozone water directly correlate with increase in the ozone decay time in water after lowering the pH value of water from pH ¼ 7 to 4 indicating that acidic ozone water is an effective means of sterilizing microbes. However, advanced cells such as fertilized eggs were not greatly influenced by the acidic ozone water. Also, both human and canine cells after treatment with the acidic ozone water prospered without showing signs of stress due to ozone in acidic water. This study suggests that antioxidant enzymes such as superoxide dismutase can be developed in the advanced cells to protect themselves from attacks by reactive oxygen species. Meanwhile, the advanced cells utilize oxygen by certain enzymes, proliferating life on earth. Ó 2013 Elsevier B.V. All rights reserved.
Keywords: Reactive oxygen species Sterilization of microbes Antioxidant Ozone
1. Introduction The influence of reactive oxygen species (ROS) on living cells including various microbes is a very important issue at present. Multicelluar organisms can produce ROS in response to infection and cellular stress, or as a result of oxidative metabolism of nutrients. ROS then can be used for host defense against invading pathogens, and to modulate cell signaling pathways affecting immune responses, death and proliferation of cells, and metabolism. In spite of such broad and important roles of ROS, the fundamental mechanism by which ROS modulate cellular processes is not well understood in part due to diffusive nature of small molecules and rapid conversion of ROS to stable molecules. The ROS includes excited oxygen atoms (O*), super oxides (O 2 ), ozone (O3), nitrogen monoxide (NO), and hydrogen peroxide (H2O2). Therefore, understanding the biological effects of individual component of ROS in well-defined model systems would help delineating the mechanism of action of ROS. The jet configuration used for the generation of atmospheric non-thermal plasmas [1] has become one of the key devices for attaining ROS by stable plasma for biological applications. The reactive species produced from these plasma jets are blown out to
* Corresponding author. E-mail address: [email protected] (H.S. Uhm). 1567-1739/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cap.2012.12.018
a separate region for surface modification, thus obtaining plasma stability and active biological and chemical reactions of the plasmas. Such plasma sources provide a convenient means of sterilization, surface functionalization, cell removal, and the micro-contact printing of proteins onto polymer substrates in biological applications [2]. To satisfy the requirements of these diverse applications, many electrical discharge configurations have been proposed and studied. One of the promising sources is the Atmospheric Pressure Plasma Jet (APPJ), which produces short-lived reactive species and propels them out of the source [3]. Another commonly used ROS is ozone, which is very effective for sterilizing microbes. Ozone after sterilization disintegrates into oxygen without conveying any harmful materials into the environment. The difficulties associated with ozone are its finite lifetime in water and its low efficiency. In this context, we investigated the influence of excited oxygen from a plasma jet and ozone in acidic water on the efficacy of sterilization of microbes and on the survival of mammalian cells.
2. Methods and results 2.1. Sterilization of microbes by an RF plasma jet Electrodes consisting of coaxial cylinders are usually used with the APPJ, and the stable discharge is realized by cooling both
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electrodes and increasing the flow rate of the gas. The reactor of the plasma jet consists of two coaxial cylindrical electrodes. The inner electrode has a stepped structure which is partially covered by a dielectric tube at the top of the electrode and is connected to a 13.56 MHz radio-frequency generator through a matching network. The outer electrode is grounded and encloses the inner electrode. Fig. 1 shows the optical emission spectra of Ar/O2 and He/O2 plasma jets in the range of 650e850 nm. The emission intensity of the Ar/O2 plasma jet flame is significantly higher than that of the He/O2 plasma flame over the entire range. Specifically, the plasma flame produced by the Ar/O2 plasma jet has a relatively high emission intensity of exited oxygen atoms, of which lines existing at 777.4 nm (3p5P / 3s5S) and 844.6 nm (3p3P / 3s3S) stem from the dissociative excitation and the direct excitation processes. The effects of oxygen radicals in the Ar/O2 plasma jet on sterilization were actinometrically measured in terms of the O2 concentration using the emission intensity ratio of O* (844.6 nm) and Ar* (750.4 nm) [4e6]. Although the discharge characteristics of an argoneoxygen plasma jet were recently investigated [7,8], this type of jet is nonetheless unstable and difficult to maintain when oxygen gas is added for practical applications. However, a stepped-electrode scheme [9] resolved the issues associated with operation of the argon plasma jet with negative gases such as oxygen. The sterilization of Bacillus atrophaeus spores in argoneoxygen plasma jets was conducted [10,11], with the results showing that the argon plasma jet expends not only a small amount of relatively inexpensive argon gas and consumes less power, but also that it requires a relatively short treatment time compared to a helium plasma jet, even with a low flame temperature. The experimental data showed that the sterilizing efficiency of the argoneoxygen plasma jet is much better than that of the heliumeoxygen plasma jet. Previous studies [8,11] indicated that the electron density in an argon plasma jet under identical electrical discharge conditions is higher than that in a helium plasma jet. To identify the relationship between the sterilization effects and the O2 concentration in an atmospheric-pressure Ar/O2 plasma jet, the sterilization efficacy with the O2 concentration was measured by colony counts of surviving microbes and the result was compared with the emission intensity ratios of O* (844.6 nm) and Ar* (750.4 nm) [10], showing that the highest emission intensity ratio of O* (844.6 nm) and Ar* (750.4 nm) occurs at the specified O2 concentration corresponding to optimum sterilization efficacy.
Fig. 1. Optical emission spectra in the range of 650 nme850 nm: Ar/O2 plasma jet, and He/O2 plasma jet with electric power of 130 W and a feed gas flow of 10 lpm for Ar and He, respectively, added to 25 ccm of O2 (0.25 vol.%).
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Fig. 2 shows the morphological change of B. atrophaeus spores as imaged by SEM (JSM-5410LV, JEOL). The spores images shown in Fig. 2(a)e(d) show untreated spores as well as those treated by heating gas (90 C, and 30 s), treated by Ar/O2 plasma jet (130 W, 85 C, and 20 s) and treated by Ar/O2 plasma jet (130 W, 85 C, and 30 s), respectively [11]. The heating gas and the Ar/O2 plasma jet were operated at 10 lpm of Ar and at 25 ccm of O2 (0.25 vol.%). As shown in Fig. 2(b), B. atrophaeus spores treated by heating gas show no changes compared to the untreated spores in Fig. 2(a). In contrast with Fig. 2(a) and (b), the spores treated by the Ar/O2 plasma jet show a distinctly changed morphology. The size and number of spores in Fig. 2(c) and (d) were both reduced as the treatment time increased. We therefore conclude that the decrease in the spore size is related to the oxidation of the spores and that the Ar/O2 plasma jet effectively sterilizes B. atrophaeus endospores using reactive oxygen species. The thermal effects may not be important. However, further study of the relationship between the viability and the spore size is required in the form of a microbiological study. For a further investigation of the morphological changes of microbes by a plasma treatment, transmission electron microscopy was carried out (TEM) on Escherichia coli. E. coli were grown in tryptic soy broth (TSB) for 3 h and then centrifuged at 3000 rpm for 10 min. The cells were removed to a cover glass and treated with plasma for 2 min and then returned to TSB and centrifuged for 10 min. Cells were harvested, and TSA mixed with 2.5% glutaraldehyde in 0.1 M PBS was used to fix the agar block. Samples were post-fixed in 1% (w/v) OsO4. The fixed samples were placed in 0.1 M PBS for 2 h at room temperature, washed once with the same buffer, dehydrated in a graded ethanol series, and embedded in a lowviscosity medium. Thin sections of the specimens were cut with a diamond knife on an Ultracut Ultramicrotome (Super Nova; Reichert-Jung Optische Werke, Wien, Austria), and sections were double-stained with uranyl acetate and lead citrate. The grids were examined with an H-7000FA transmission electron microscope (Hitachi) at an operating voltage of 75 kV. Transmission electron micrographs [12] showed morphological changes in E. coli cells treated by atmospheric plasma at 75 W for 2 min (Fig. 3). The treated cells showed severe cytoplasmic deformation and leakage of their cellular components, including the bacterial chromosome. The results clearly demonstrate the loss of viability of bacterial cells after the plasma treatment. 2.2. Sterilization of microbes using acidic ozone water Biological warfare agents such as viruses or bacteria can attach themselves to organic or inorganic aerosols and spread when the aerosol particles float around, eventually settling on the surfaces of various objects with abundant organic compounds. Most of the ozone molecules in acidic ozone water disappear due to interaction with organic compounds in the vicinity of microbes. Only a fraction of ozone in acidic ozone water participates in the killing activity of biological warfare agents. In this context, the ozone concentration in acidic ozone water must be considerably higher than what would be expected and the pH value of the acidic water must be significantly lower for the sterilization of areas contaminated with biological warfare agents [13]. In other words, the ozone decay time s and killing rate associated with acidity in an environment containing abundant organic compounds in the real world are much lower than the expected values in a controlled experiment without organic contamination. Acidic ozone water can be sprayed over a large surface area contaminated with biological warfare agents. The acidic ozone water can also effectively kill other ordinary microbes [14] of viruses, bacteria and fungi, making it applicable to the agriculture, seafood and livestock industries for the preservation of various products while also being useful in hospitals or other
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Fig. 2. Scanning electron micrographs (10,000) of B. atrophaeus spores: (a) untreated, (b) treated with heating gas (90 C, 30 s), (c) treated with the Ar/O2 plasma jet (85 C, 20 s), and (d) treated with the Ar/O2 plasma jet (85 C, 30 s). The atmospheric-pressure Ar/O2 plasma jet was operated at 130 W, 0.25 vol.% O2, and with an exposure distance of 0.5 cm.
germ-infested areas as a disinfectant. Furthermore, the ozone in acidic seawater [15] sterilizes very effectively a large amount of seawater in a short time. Acidic water is made from neutral water that is mixed acidic materials. Mixing a small amount of acid such as hydrochloric acid (HCl) into water produces acidic water [13]. The acidity of acidic water is represented by its pH value. Neutral fresh water [13] has a pH value of 7. On the other hand, natural seawater is slightly alkaline [15] and has a pH value of 8.2. The acidity of acidic water increases as the pH value is lowered from 7 for fresh water or from 8.2 for seawater. The pH value of acidic water was measured here via the mixing ratio of the hydrochloric acid used to create it. Fig. 4 shows plots of the pH value versus the concentration of the hydrochloric acid in units of milli-mole per liter (mM/L) for three different types of water [13,15], in this case deionized water, tap water supplied from a municipal water supply system, and seawater. The square dots represent the acidity of the acidic water from the deionized water, the circular dots represent the acidity of the acidic water made from the tap water, and the triangular dots represent the acidity of the acidic seawater. Note in Fig. 4 that the pH value of the neutral seawater without concentrated acid is pH ¼ 8.2. The pH values of the acidic waters shown in Fig. 4 are reduced as the concentration of the hydrochloric acid increases, thereby enhancing the acidity. The pH value in the acidic seawater [15] has very peculiar profile in terms of the concentration of the hydrochloric acid. This peculiar property may be caused by the various ions existing in the seawater, including sodium and chlorine ions. One ton of acidic water with a pH value of 4 made from tap water as represented by the circular dots in Fig. 4 requires 0.6 mol of hydrochloric acid, which is equivalent to 22 g of the acid. Obviously, a very small amount of acid is needed to make acidic water from tap water. The acidity at a pH value of 4 is similar to the acidity of cola and is also used for baby skincare products. It was also noted from Fig. 4 that one ton of acidic seawater with a pH value of 6 made from seawater requires 1.5 mol of hydrochloric acid, which is equivalent to 55 g of the acid. Clearly, a very small
amount of acid is also needed to make acidic seawater from plain seawater. One liter of seawater contains 35 g of salt, which is equivalent to 0.54 mol. Thus, the mole fraction of the hydrochloric acid to the salt concentration in the acidic seawater with a pH of 6 is 0.0027, which is negligibly small. A corona-discharge ozone generator produces gas with a high ozone concentration which is injected into a porous ceramic diffuser submerged in acidic water to generate acidic ozone water (AOW). The ozone gas can also be dissolved in acidic water by an ozone mixture device based on the Bernoulli effect, which mixes tiny bubbles of ozone gas with the water, dissolving about 60 percent of the ozone into the water. The dissolved ozone concentration in AOW is in the range of 0.1e100 milligrams per liter (mg/L), as measured by ultraviolet spectroscopy. The focus of sterilization research [13,14] is mostly on the decontamination of bacterial endospores because they are recognized to be the most difficult microorganisms to kill. A decontamination experiment involving bacterial endospores was carried out using spores of B. atrophaeus (Bacillus subtilis var. niger, ATCC 9372). In order to observe the influence of organic compounds on the ozone concentration and to determine its kill properties, the original bacillus spore suspension [13e15] had a high concentration (40% by weight) of ethanol, which is harmless to spores. The spore concentration of the original spore suspension was 107e108 spores per milliliter (mL). The spore-treatment experiments were conducted by adding 0.1 mL of spore suspension to 10 mL of acidic ozone water with three different pH values of 4, 5 and 7. The acidic ozone water in this example was made of tap water supplied from a municipal water supply system. The ozone concentration in the AOW was 20 mg/L. The concentration of ethanol in 10 mL of AOW was calculated to be 0.072 mol/L. Ozone in water decayed very rapidly with this ethanol concentration. One milliliter of the solution was obtained from each sample after a specified contact time and was diluted with 9 mL of distilled water. Fig. 5 shows the survival curves [13] for B. atrophaeus endospores exposed to the bactericidal formulation, AOW, with pH
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Fig. 5. Plots of the survival curves for B. atrophaeus endospores exposed to the bactericidal formulation, AOW, with pH values of 4, 5, and 7 for an ethanol concentration of 0.072 mol/L. The vertical axis is the log of the ratio of the number of viable spores remaining (N) to the control number of N0. Dots are experimental data and curves are obtained theoretically from Eq. (1).
Fig. 3. Transmission electron microscopy of E. coli (4400): (a) control cells, (b) cells after 2 min of plasma treatment at 75 W.
values of 4, 5, and 7 for an ethanol concentration of 0.072 mol/L. The vertical axis is the log of the ratio of the number of viable spores remaining (N) to the control number of N0. Each point in Fig. 5 represents an average value of three data measurements. An untouched control was also analyzed every time to obtain the average control number N0 ¼ 2.5 106, corresponding to log N0 ¼ 6.4. The error bars in Fig. 5 were obtained from the square root of the second moment of data around its mean value at each contact time. The ozone in the acidic ozone water decayed faster within 1 min with the decay time being less than 30 s due to the ethanol contamination. Therefore, most of the killing action in the acidic ozone water occurred within 1 min, as expected. Keeping in mind N0 ¼ 2.5 106, it should be noted that most of the spores were killed within 2 min by contact with the acidic ozone water at a low pH value. The curves in Fig. 5 represent the log reduction of live microbes versus the time t in seconds for the acidic ozone water [13,14], obtained from
DIW Tap Water Seawater
8 7
pH
6 5 4 3 0
1
2
3
4
Hydrochloric Acid (mM/L) Fig. 4. Plots of the pH value versus the concentration of the hydrochloric acid in units of milli-mole per liter (mM/L) for the following three different types of waters: deionized water, tap water supplied from a municipal water supply system, and seawater. The square dots represent the acidity of the acidic water created from the deionized water, the circular dots represent the acidity of the acidic water made from the tap water, and the triangular dots represent the acidity of the acidic seawater.
log
NðtÞ N0
¼ 0:43anO3 s½1 exp ðt=sÞ;
(1)
where the constant N0 represents the initial density of the microorganisms, nO3 is the initial ozone density, and a is the inactivation coefficient of ozone in units of L/(mg s). The microbe number N in Eq. (1) in the unit volume can be estimated from Eq. (1) in terms of the time t. The curves in Fig. 5 were obtained from Eq. (1) for AOW, with nO3 ¼ 20 mg/L, s ¼ 8.1 s for pH ¼ 7, s ¼ 23 s for pH ¼ 5 and s ¼ 26 s for pH ¼ 4. These ozone decay times were measured values at an ethanol concentration of 0.072 mol/L. The parameter a ¼ 0.0215 L/(mg s) when obtaining the curves [13] here was the least-squares value fitted to the experimental data (triangular dots) for pH ¼ 7 in Fig. 5. Note that the ozone decay time s increases from s ¼ 8.1 s for pH ¼ 7 to s ¼ 23 s for pH ¼ 5 and to s ¼ 26 s for pH ¼ 4 in AOW at room temperature (25 C). The short decay time s ¼ 8.1 s in AOW at pH ¼ 7 is for a situation in which the environment contains many organic compounds, represented by an ethanol concentration of 0.072 mol/L corresponding to 3.4 g/L. The ozone concentration of 20 mg/L is far less than the ethanol concentration. However, the 20 mg/L ozone concentration is equivalent to 1.4 1017 molecules/cm3, which is much higher than the spore concentration on the order of 106/cm3. It is shown in Fig. 5 that the log of the ratio of
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N to N0 for the acidic ozone water in the experimental data agrees remarkably well with the theoretical curves. The acidic ozone water may be also very effective for the inactivation of H1N1 viruses [16], also known as the swine flu virus, which appeared almost daily in the news during the summer of 2009 and which is a highly contagious disease. The sterilization of microbes in seawater [15] was carried out using ozone and acid in seawater. The spore concentration of the original spore suspension was 105e106 spores per mL. The sporetreatment experiments were conducted by adding 0.2 mL of the spore suspension to 10 mL of seawater at a specified pH value and at an ozone concentration of 5 mg/L. The concentration of ethanol in the mixture of 0.1 mL of the spore suspension and 10 mL of the seawater was 7.7 mM/L. Ozone in the water decayed rapidly at this ethanol concentration [15]. For example, the ozone decay time s in the seawater with an ethanol concentration of 7.7 mM/L was measured to be s ¼ 22 s for pH ¼ 8, s ¼ 40 s for pH ¼ 7, s ¼ 70 s for pH ¼ 6 and s ¼ 90 s for pH ¼ 5. One milliliter of the solution was obtained from each sample after a contact time of 40 min and was diluted with 9 mL of distilled water. The contact time t ¼ 40 min is much longer than the ozone decay time s of less than 2 min with a high concentration of ethanol. Eq. (1) is further simplified to [15]
log
NðtÞ ¼ 0:43anO3 s N0
(2)
for t >> s, which is typical for the sterilization of microbes in seawater. Fig. 6 shows the survival curve [15] for B. atrophaeus endospores exposed to seawater with a 5 mg/L ozone concentration at several different pH values. The horizontal scale represents the ozone decay time s measured in seconds corresponding to the specific pH value of seawater contaminated by an ethanol concentration of 7.7 mM/L. The vertical axis is the log of the ratio of the number of viable spores remaining (N) to the control number of N0. An untouched control was also analyzed each time to obtain the average control number N0 ¼ 3.3 105, which corresponds to log N0 ¼ 5.52. The dots in Fig. 6 represent the experimental data [15] pertaining to the log reduction of live microbes versus the ozone decay time s in seconds for seawater with an ozone concentration of 5 mg/L contaminated by an ethanol concentration of 7.7 mM/L corresponding to 360 mg/L. In effect, all of the spores were killed at
s ¼ 90 s, but one surviving spore at s ¼ 90 s was assumed for convenience regarding the log-scale plot shown in Fig. 6. The molecular number of ethanol in this seawater is 150 times greater than that of ozone. The straight line in Fig. 6 was obtained from Eq. (3) and was linearly fitted to the experimental dots (squares) with the parameter anO3 ¼ 0.135/s, which was the least-squares value fitted to the experimental data in Fig. 6. Assuming an initial ozone concentration of nO3 ¼ 5 mg/L, the inactivation coefficient [15] of ozone was calculated to be a ¼ 0.027 L/(mg s) for anO3 ¼ 0.135/s. The short decay time s in Fig. 6 represents a situation in which the seawater contains many organic compounds. On the other hand, for relatively clean seawater in an application to ballast water, the organic material is less than 5 mg/L and the ozone decay time at pH ¼ 7 is 3.3 min. Eq. (3) predicts the viable Bacillus spore number of N ¼ 8 for N0 ¼ 3.3 105 at an ozone concentration of nO3 ¼ 2 mg/L. Fig. 6 shows that the log of the ratio of N to N0 for the acidic seawater in the experimental data is in good agreement with the theoretical model. The ozone decay time of s ¼ 90 s at pH of 5 is four times that at a pH of 8. Therefore, an increase of the ozone decay time by lowering the pH value must play a pivotal role in the killing process. Similar sterilization can be achieved by a four-fold increase in the ozone concentration in seawater at pH ¼ 8. However, an ozone concentration of 20 mg/L in seawater may be impractical for sterilization applications. Hence, a reasonable ozone concentration at a low pH value may make it possible to sterilize a large amount of seawater in relatively little time, freeing this water from unwanted microbes. Fig. 6 clearly demonstrates that an increase of the ozone decay time at a low pH has the most important synergic effect on the sterilization of microbes in seawater. Other studies also show that acidic ozone water is a very effective means of sterilizing vegetative bacteria and viruses. Particularly, these studies indicated that acidic ozone water effectively disinfected enveloped and naked viruses in environments contaminated with numerous organic compounds. The acidic ozone water kills more than 99.98% of viruses, except for the CoxB3 virus [14]. Therefore, it is a good disinfectant of viruses. The HIV virus is more easily killed [14] than the FluA virus, although its death profile is similar to that of the FluA virus. Acidic ozone water may also be useful for disinfection in hospitals or other germ-infested areas. Finally, it is also concluded that acidic ozone water is applicable to the agricultural, seafood, and livestock industries to prevent the spoilage of products and to prolong the shelf lives of these products. 3. Discussion
Fig. 6. Plots of the survival curve for B. atrophaeus endospores exposed to seawater with a 5 mg/L ozone concentration at several different pH values. The horizontal scale represents the ozone decay time s measured in seconds corresponding to the specific pH value of seawater contaminated by an ethanol concentration of 7.7 mM/L.
According to the findings as given above, reactive oxygen species, particularly excited oxygen atoms and ozone molecules, are very effective for the sterilization of microbes. This leads to the question of what effects ROS (for example, acidic ozone water) has on advanced cells. To test this, an experiment was conducted on fertilized eggs. We injected tap water into 10 fertilized eggs, finding that all of these eggs were dead a few days after the water injection. It is likely that E. coli existing in the tap water may have caused the death of these eggs. We also injected acidic ozone water made of this tap water into 10 fertilized eggs. Remarkably, all of the eggs survived without any harm from the injection of the acidic ozone water. The E. coli typically existing in tap water was most likely eliminated by the acidic ozone water, which was then not harmful to the fertilized eggs. Thus, the ozone in the water may not be critically harmful to advanced living cells. A second experiment was related to the inactivation experiment involving the viruses. All viruses need a host to survive. Therefore, we used human cells or canine cells as the host cells for the viruses [14]. Therefore, we were forced to evaluate the survivability of the host cells in the stressful
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condition of the acidic ozone water, finding that all of the host cells survived well due to the treatment with acidic ozone water in a physical parameter range in which other microbes are destroyed. According to our previous observations of various microbes, the spore walls of B. atrophaeus (B. subtilis var. niger, ATCC 9372) are well coated with protein, whereas human or canine cells have membrane walls that are much more fragile than the Bacillus spore wall. Yet, canine cells are more resilient than Bacillus spore. Why are advanced cells such as canine cells more resilient than microbes in the acidic ozone water? We speculate that advanced cells have a variety of antioxidant enzymes, such as superoxide dismutase, which may protect the cells from the toxicity of the acidic ozone water. Hence, human cells may experience minimal damage from acidic ozone water at an intensity level that would otherwise exert a devastating effect on microbes. Reactive oxygen species including O*, O 2 , O3, NO, and H2O2 may have appeared when free oxygen became available in the earth’s atmosphere. The newly born cells in an oxygen-rich atmosphere may have developed a defense system against the toxicity of ROS. Enzymes such as superoxide dismutase may exist in these advanced cells, offering them protection against the threat of ROS toxicity. Meanwhile, these advanced cells may develop the capability of oxygen utilization by making use of certain enzymes, invigorating their activity and allowing this species to proliferate on earth. We would like to know what portion of DNA is responsible for the development of the antioxidant enzymes and what portion of DNA is responsible for the enzyme development as it pertains to oxygen utilization. Based on current evidence of volcanic activity, the first atmosphere on earth would have contained 60% hydrogen, 20% oxygen in the form of water vapor, 10% carbon dioxide, and 7% hydrogen sulfide. Major rainfall events led to the buildup of a vast ocean, enriching the other agents such as carbon dioxide, nitrogen and inert gases. The second atmosphere started about 3.4 billion years ago and mostly consisted of nitrogen. Hints of early life forms were found around this time period [17]. An oxygen-containing atmosphere began to develop about 1.7 billion years ago in the late Archaean eon, although the oxygen content was tenuous. Evidence of a third atmosphere can be seen with the development of red beds and the end of the banded iron formations, signifying a shift from a reducing atmosphere to an oxidizing atmosphere. Most free oxygen (O2) arose about 650 million years ago, fluctuating in its concentration, up and down, until it reached a steady state of more than 15 percent. Following this time span was the Phanerozoic eon, during which oxygen-breathing metazoan life forms began to appear. Meanwhile, simple cells such as prokaryotes appeared on earth around 3.8 billion years ago [18]. Complex cells in eukaryotes started to show up about 2 billion years ago. Multicellular life was initiated around 1 billion years ago. Advanced cells such as simple animals appeared on earth 600 million years ago, which coincides with the appearance of most of the free oxygen in the third atmosphere. Life on earth proliferated drastically about 600 million years ago due to the drastic increase in the oxygen content in the atmosphere, known as called Cambrian explosion. The accumulation of atmospheric oxygen allowed the formation of an ozone layer. This began to block ultraviolet radiation, permitting the colonization of the land. This suggests that advanced cells developed their capability to utilize oxygen by certain enzymes while protecting themselves from the toxicity of reactive oxygen species. 4. Conclusion A sterilization experiment with bacterial endospores indicates that an argoneoxygen plasma jet very effectively kills endospores
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of B. atrophaeus (ATCC 9372), thereby demonstrating its capability to clean surfaces and its usefulness for reinstating contaminated equipment as free from toxic biological agents. The key element of the sterilization process is the oxygen radicals. Transmission electron micrographs showed morphological changes in E. coli cells treated with atmospheric plasma at 75 W for 2 min. The treated cells showed severe cytoplasmic deformation and leakage of the bacterial chromosome material. The results clearly demonstrate the loss of viability of bacterial cells after the plasma treatment. Ozone is one of the most reactive oxygen species. Due to the synergic effects of the acidity and ozone, acidic ozone water is a very effective means of sterilizing various microbes. However, advanced cells such as human cells survive reasonably well after a treatment of acidic ozone water in the physical parameter range in which other microbes are devastated. This finding suggests that the enzymes such as superoxide dismutase may exist in these advanced cells, protecting them from the threat of ROS toxicity while these advanced cells develop the capability to utilize oxygen by making use of certain enzymes, invigorating their activity levels and allowing their species to proliferate on earth. This observation leads to many questions to be answered in future studies. The questions are as follow: How do antioxidant enzymes such as superoxide dismutase protect cells from the toxicity of ROS? Do other components of ROS show similar killing efficacy as ozone in acidic water? When do these enzymes appear in living cells on earth? Can we establish a timeline relationship between the evolution of living cells and the evolution of the atmosphere on earth? Can we find a relationship between ROS and the ROS defense system by making use of our ROS from plasma and ozone? We must design specific experimental procedures to answer all of these questions in future studies. Acknowledgements This work was financially supported by the SRC program (Grant #2010-0029421) of the National Research Foundation (NRF) and was also partially supported by the Research Grant of Kwangwoon University in 2013, and program funds from the Western Human Nutrition Research Center/ARS/USDA (USDA is an equal opportunity employer and provider). References [1] Y.C. Hong, H.S. Uhm, W.J. Yi, Appl. Phys. Lett. 93 (2008) 051504. [2] K.H. Becker, U. Kogelschatz, K.H. Schoenbach, R.J. Barker, Non-equilibrium Air Plasmas at Atmospheric Pressure, Institute of Physics, Bristol, 2005. [3] J. Park, I. Henins, H.W. Herrmann, G.S. Selwyn, R.F. Hicks, J. Appl. Phys. 89 (2001) 20. [4] M.A. Lieberman, A.J. Lichtenberg, Principles of Plasma Discharges and Materials Processing, second ed., Wiley, New York, 2005, pp. 270e279. [5] R.E. Walkup, K.L. Saenger, G.S. Selwyn, J. Chem. Phys. 84 (1986) 2668. [6] D. Pagnon, J. Amorim, J. Nahorny, M. Touzeou, M. Vialle, J. Phys. D 28 (1995) 1856. [7] S. Wang, V. Schulz-von der Garhen, H.F. Dobele, Appl. Phys. Lett. 83 (2003) 3272. [8] S.Z. Li, J.P. Lim, J.G. Kang, Han. S. Uhm, Phys. Plasmas 13 (2006) 093503. [9] J.P. Lim, H.S. Uhm, S.Z. Li, Appl. Phys. Lett. 90 (2007) 051504. [10] H.S. Uhm, J.P. Lim, S.Z. Li, Appl. Phys. Lett. 90 (2007) 261501. [11] J.P. Lim, H.S. Uhm, S.Z. Li, Phys. Plasmas 14 (2007) 093504. [12] Y.F. Hong, J.G. Kang, H.Y. Lee, H.S. Uhm, E. Moon, Y.H. Park, Lett. Appl. Microbiol. 48 (2009) 33. [13] H.Y. Lee, H.S. Uhm, Y.F. Hong, Y.H. Park, Appl. Phys. Lett. 92 (2008) 174102. [14] H.S. Uhm, H.Y. Lee, Y.C. Hong, D.H. Shin, Y.H. Park, Y.F. Hong, C.K. Lee, J. Appl. Phys. 102 (2007) 013303. [15] H.S. Uhm, Y.F. Hong, H.Y. Lee, Y.H. Park, J. Korean Phys. Soc. 56 (2010) 108. [16] H.S. Uhm, K.H. Lee, B.L. Seong, Appl. Phys. Lett. 95 (2009) 173704. [17] J. Schopf, Earth’s Earliest Biosphere: Its Origin and Evolution, Princeton University Press, Princeton, N.J, 1983. [18] Carl Woese, J. Peter Gogarten, When did eukaryotic cells (cells with nuclei and other internal organelles) first evolve? What do we know about how they evolved from earlier life-forms? Sci. Am. (October 21, 1999).
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Current Applied Physics journal homepage: www.elsevier.com/locate/cap
Influence of reactive oxygen species on the sterilization of microbes Han S. Uhm a, *, Eun H. Choi a, Guang S. Cho a, Daniel H. Hwang b a
Department of Electrophysics, Kwangwoon University, 447-1 Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of Korea Department of Nutrition and Western Human Nutrition Research Center, USDA ARS, University of California, One Shields Avenue, Davis, CA 95616-5270, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2012 Received in revised form 12 December 2012 Accepted 29 December 2012 Available online 7 January 2013
The influence of reactive oxygen species on living cells, including various microbes, is discussed. A sterilization experiment with bacterial endospores reveals that an argoneoxygen plasma jet very effectively kills endospores of Bacillus atrophaeus (ATCC 9372), thereby indicating that oxygen radicals are the key element of sterilization. Ozone in acidic water also kills endospores of B. atrophaeus very effectively, demonstrating the capability of cleaning a large surface area contaminated by toxic biological agents. The viable microbe numbers after the contact with acidic ozone water directly correlate with increase in the ozone decay time in water after lowering the pH value of water from pH ¼ 7 to 4 indicating that acidic ozone water is an effective means of sterilizing microbes. However, advanced cells such as fertilized eggs were not greatly influenced by the acidic ozone water. Also, both human and canine cells after treatment with the acidic ozone water prospered without showing signs of stress due to ozone in acidic water. This study suggests that antioxidant enzymes such as superoxide dismutase can be developed in the advanced cells to protect themselves from attacks by reactive oxygen species. Meanwhile, the advanced cells utilize oxygen by certain enzymes, proliferating life on earth. Ó 2013 Elsevier B.V. All rights reserved.
Keywords: Reactive oxygen species Sterilization of microbes Antioxidant Ozone
1. Introduction The influence of reactive oxygen species (ROS) on living cells including various microbes is a very important issue at present. Multicelluar organisms can produce ROS in response to infection and cellular stress, or as a result of oxidative metabolism of nutrients. ROS then can be used for host defense against invading pathogens, and to modulate cell signaling pathways affecting immune responses, death and proliferation of cells, and metabolism. In spite of such broad and important roles of ROS, the fundamental mechanism by which ROS modulate cellular processes is not well understood in part due to diffusive nature of small molecules and rapid conversion of ROS to stable molecules. The ROS includes excited oxygen atoms (O*), super oxides (O 2 ), ozone (O3), nitrogen monoxide (NO), and hydrogen peroxide (H2O2). Therefore, understanding the biological effects of individual component of ROS in well-defined model systems would help delineating the mechanism of action of ROS. The jet configuration used for the generation of atmospheric non-thermal plasmas [1] has become one of the key devices for attaining ROS by stable plasma for biological applications. The reactive species produced from these plasma jets are blown out to
* Corresponding author. E-mail address: [email protected] (H.S. Uhm). 1567-1739/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cap.2012.12.018
a separate region for surface modification, thus obtaining plasma stability and active biological and chemical reactions of the plasmas. Such plasma sources provide a convenient means of sterilization, surface functionalization, cell removal, and the micro-contact printing of proteins onto polymer substrates in biological applications [2]. To satisfy the requirements of these diverse applications, many electrical discharge configurations have been proposed and studied. One of the promising sources is the Atmospheric Pressure Plasma Jet (APPJ), which produces short-lived reactive species and propels them out of the source [3]. Another commonly used ROS is ozone, which is very effective for sterilizing microbes. Ozone after sterilization disintegrates into oxygen without conveying any harmful materials into the environment. The difficulties associated with ozone are its finite lifetime in water and its low efficiency. In this context, we investigated the influence of excited oxygen from a plasma jet and ozone in acidic water on the efficacy of sterilization of microbes and on the survival of mammalian cells.
2. Methods and results 2.1. Sterilization of microbes by an RF plasma jet Electrodes consisting of coaxial cylinders are usually used with the APPJ, and the stable discharge is realized by cooling both
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electrodes and increasing the flow rate of the gas. The reactor of the plasma jet consists of two coaxial cylindrical electrodes. The inner electrode has a stepped structure which is partially covered by a dielectric tube at the top of the electrode and is connected to a 13.56 MHz radio-frequency generator through a matching network. The outer electrode is grounded and encloses the inner electrode. Fig. 1 shows the optical emission spectra of Ar/O2 and He/O2 plasma jets in the range of 650e850 nm. The emission intensity of the Ar/O2 plasma jet flame is significantly higher than that of the He/O2 plasma flame over the entire range. Specifically, the plasma flame produced by the Ar/O2 plasma jet has a relatively high emission intensity of exited oxygen atoms, of which lines existing at 777.4 nm (3p5P / 3s5S) and 844.6 nm (3p3P / 3s3S) stem from the dissociative excitation and the direct excitation processes. The effects of oxygen radicals in the Ar/O2 plasma jet on sterilization were actinometrically measured in terms of the O2 concentration using the emission intensity ratio of O* (844.6 nm) and Ar* (750.4 nm) [4e6]. Although the discharge characteristics of an argoneoxygen plasma jet were recently investigated [7,8], this type of jet is nonetheless unstable and difficult to maintain when oxygen gas is added for practical applications. However, a stepped-electrode scheme [9] resolved the issues associated with operation of the argon plasma jet with negative gases such as oxygen. The sterilization of Bacillus atrophaeus spores in argoneoxygen plasma jets was conducted [10,11], with the results showing that the argon plasma jet expends not only a small amount of relatively inexpensive argon gas and consumes less power, but also that it requires a relatively short treatment time compared to a helium plasma jet, even with a low flame temperature. The experimental data showed that the sterilizing efficiency of the argoneoxygen plasma jet is much better than that of the heliumeoxygen plasma jet. Previous studies [8,11] indicated that the electron density in an argon plasma jet under identical electrical discharge conditions is higher than that in a helium plasma jet. To identify the relationship between the sterilization effects and the O2 concentration in an atmospheric-pressure Ar/O2 plasma jet, the sterilization efficacy with the O2 concentration was measured by colony counts of surviving microbes and the result was compared with the emission intensity ratios of O* (844.6 nm) and Ar* (750.4 nm) [10], showing that the highest emission intensity ratio of O* (844.6 nm) and Ar* (750.4 nm) occurs at the specified O2 concentration corresponding to optimum sterilization efficacy.
Fig. 1. Optical emission spectra in the range of 650 nme850 nm: Ar/O2 plasma jet, and He/O2 plasma jet with electric power of 130 W and a feed gas flow of 10 lpm for Ar and He, respectively, added to 25 ccm of O2 (0.25 vol.%).
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Fig. 2 shows the morphological change of B. atrophaeus spores as imaged by SEM (JSM-5410LV, JEOL). The spores images shown in Fig. 2(a)e(d) show untreated spores as well as those treated by heating gas (90 C, and 30 s), treated by Ar/O2 plasma jet (130 W, 85 C, and 20 s) and treated by Ar/O2 plasma jet (130 W, 85 C, and 30 s), respectively [11]. The heating gas and the Ar/O2 plasma jet were operated at 10 lpm of Ar and at 25 ccm of O2 (0.25 vol.%). As shown in Fig. 2(b), B. atrophaeus spores treated by heating gas show no changes compared to the untreated spores in Fig. 2(a). In contrast with Fig. 2(a) and (b), the spores treated by the Ar/O2 plasma jet show a distinctly changed morphology. The size and number of spores in Fig. 2(c) and (d) were both reduced as the treatment time increased. We therefore conclude that the decrease in the spore size is related to the oxidation of the spores and that the Ar/O2 plasma jet effectively sterilizes B. atrophaeus endospores using reactive oxygen species. The thermal effects may not be important. However, further study of the relationship between the viability and the spore size is required in the form of a microbiological study. For a further investigation of the morphological changes of microbes by a plasma treatment, transmission electron microscopy was carried out (TEM) on Escherichia coli. E. coli were grown in tryptic soy broth (TSB) for 3 h and then centrifuged at 3000 rpm for 10 min. The cells were removed to a cover glass and treated with plasma for 2 min and then returned to TSB and centrifuged for 10 min. Cells were harvested, and TSA mixed with 2.5% glutaraldehyde in 0.1 M PBS was used to fix the agar block. Samples were post-fixed in 1% (w/v) OsO4. The fixed samples were placed in 0.1 M PBS for 2 h at room temperature, washed once with the same buffer, dehydrated in a graded ethanol series, and embedded in a lowviscosity medium. Thin sections of the specimens were cut with a diamond knife on an Ultracut Ultramicrotome (Super Nova; Reichert-Jung Optische Werke, Wien, Austria), and sections were double-stained with uranyl acetate and lead citrate. The grids were examined with an H-7000FA transmission electron microscope (Hitachi) at an operating voltage of 75 kV. Transmission electron micrographs [12] showed morphological changes in E. coli cells treated by atmospheric plasma at 75 W for 2 min (Fig. 3). The treated cells showed severe cytoplasmic deformation and leakage of their cellular components, including the bacterial chromosome. The results clearly demonstrate the loss of viability of bacterial cells after the plasma treatment. 2.2. Sterilization of microbes using acidic ozone water Biological warfare agents such as viruses or bacteria can attach themselves to organic or inorganic aerosols and spread when the aerosol particles float around, eventually settling on the surfaces of various objects with abundant organic compounds. Most of the ozone molecules in acidic ozone water disappear due to interaction with organic compounds in the vicinity of microbes. Only a fraction of ozone in acidic ozone water participates in the killing activity of biological warfare agents. In this context, the ozone concentration in acidic ozone water must be considerably higher than what would be expected and the pH value of the acidic water must be significantly lower for the sterilization of areas contaminated with biological warfare agents [13]. In other words, the ozone decay time s and killing rate associated with acidity in an environment containing abundant organic compounds in the real world are much lower than the expected values in a controlled experiment without organic contamination. Acidic ozone water can be sprayed over a large surface area contaminated with biological warfare agents. The acidic ozone water can also effectively kill other ordinary microbes [14] of viruses, bacteria and fungi, making it applicable to the agriculture, seafood and livestock industries for the preservation of various products while also being useful in hospitals or other
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Fig. 2. Scanning electron micrographs (10,000) of B. atrophaeus spores: (a) untreated, (b) treated with heating gas (90 C, 30 s), (c) treated with the Ar/O2 plasma jet (85 C, 20 s), and (d) treated with the Ar/O2 plasma jet (85 C, 30 s). The atmospheric-pressure Ar/O2 plasma jet was operated at 130 W, 0.25 vol.% O2, and with an exposure distance of 0.5 cm.
germ-infested areas as a disinfectant. Furthermore, the ozone in acidic seawater [15] sterilizes very effectively a large amount of seawater in a short time. Acidic water is made from neutral water that is mixed acidic materials. Mixing a small amount of acid such as hydrochloric acid (HCl) into water produces acidic water [13]. The acidity of acidic water is represented by its pH value. Neutral fresh water [13] has a pH value of 7. On the other hand, natural seawater is slightly alkaline [15] and has a pH value of 8.2. The acidity of acidic water increases as the pH value is lowered from 7 for fresh water or from 8.2 for seawater. The pH value of acidic water was measured here via the mixing ratio of the hydrochloric acid used to create it. Fig. 4 shows plots of the pH value versus the concentration of the hydrochloric acid in units of milli-mole per liter (mM/L) for three different types of water [13,15], in this case deionized water, tap water supplied from a municipal water supply system, and seawater. The square dots represent the acidity of the acidic water from the deionized water, the circular dots represent the acidity of the acidic water made from the tap water, and the triangular dots represent the acidity of the acidic seawater. Note in Fig. 4 that the pH value of the neutral seawater without concentrated acid is pH ¼ 8.2. The pH values of the acidic waters shown in Fig. 4 are reduced as the concentration of the hydrochloric acid increases, thereby enhancing the acidity. The pH value in the acidic seawater [15] has very peculiar profile in terms of the concentration of the hydrochloric acid. This peculiar property may be caused by the various ions existing in the seawater, including sodium and chlorine ions. One ton of acidic water with a pH value of 4 made from tap water as represented by the circular dots in Fig. 4 requires 0.6 mol of hydrochloric acid, which is equivalent to 22 g of the acid. Obviously, a very small amount of acid is needed to make acidic water from tap water. The acidity at a pH value of 4 is similar to the acidity of cola and is also used for baby skincare products. It was also noted from Fig. 4 that one ton of acidic seawater with a pH value of 6 made from seawater requires 1.5 mol of hydrochloric acid, which is equivalent to 55 g of the acid. Clearly, a very small
amount of acid is also needed to make acidic seawater from plain seawater. One liter of seawater contains 35 g of salt, which is equivalent to 0.54 mol. Thus, the mole fraction of the hydrochloric acid to the salt concentration in the acidic seawater with a pH of 6 is 0.0027, which is negligibly small. A corona-discharge ozone generator produces gas with a high ozone concentration which is injected into a porous ceramic diffuser submerged in acidic water to generate acidic ozone water (AOW). The ozone gas can also be dissolved in acidic water by an ozone mixture device based on the Bernoulli effect, which mixes tiny bubbles of ozone gas with the water, dissolving about 60 percent of the ozone into the water. The dissolved ozone concentration in AOW is in the range of 0.1e100 milligrams per liter (mg/L), as measured by ultraviolet spectroscopy. The focus of sterilization research [13,14] is mostly on the decontamination of bacterial endospores because they are recognized to be the most difficult microorganisms to kill. A decontamination experiment involving bacterial endospores was carried out using spores of B. atrophaeus (Bacillus subtilis var. niger, ATCC 9372). In order to observe the influence of organic compounds on the ozone concentration and to determine its kill properties, the original bacillus spore suspension [13e15] had a high concentration (40% by weight) of ethanol, which is harmless to spores. The spore concentration of the original spore suspension was 107e108 spores per milliliter (mL). The spore-treatment experiments were conducted by adding 0.1 mL of spore suspension to 10 mL of acidic ozone water with three different pH values of 4, 5 and 7. The acidic ozone water in this example was made of tap water supplied from a municipal water supply system. The ozone concentration in the AOW was 20 mg/L. The concentration of ethanol in 10 mL of AOW was calculated to be 0.072 mol/L. Ozone in water decayed very rapidly with this ethanol concentration. One milliliter of the solution was obtained from each sample after a specified contact time and was diluted with 9 mL of distilled water. Fig. 5 shows the survival curves [13] for B. atrophaeus endospores exposed to the bactericidal formulation, AOW, with pH
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Fig. 5. Plots of the survival curves for B. atrophaeus endospores exposed to the bactericidal formulation, AOW, with pH values of 4, 5, and 7 for an ethanol concentration of 0.072 mol/L. The vertical axis is the log of the ratio of the number of viable spores remaining (N) to the control number of N0. Dots are experimental data and curves are obtained theoretically from Eq. (1).
Fig. 3. Transmission electron microscopy of E. coli (4400): (a) control cells, (b) cells after 2 min of plasma treatment at 75 W.
values of 4, 5, and 7 for an ethanol concentration of 0.072 mol/L. The vertical axis is the log of the ratio of the number of viable spores remaining (N) to the control number of N0. Each point in Fig. 5 represents an average value of three data measurements. An untouched control was also analyzed every time to obtain the average control number N0 ¼ 2.5 106, corresponding to log N0 ¼ 6.4. The error bars in Fig. 5 were obtained from the square root of the second moment of data around its mean value at each contact time. The ozone in the acidic ozone water decayed faster within 1 min with the decay time being less than 30 s due to the ethanol contamination. Therefore, most of the killing action in the acidic ozone water occurred within 1 min, as expected. Keeping in mind N0 ¼ 2.5 106, it should be noted that most of the spores were killed within 2 min by contact with the acidic ozone water at a low pH value. The curves in Fig. 5 represent the log reduction of live microbes versus the time t in seconds for the acidic ozone water [13,14], obtained from
DIW Tap Water Seawater
8 7
pH
6 5 4 3 0
1
2
3
4
Hydrochloric Acid (mM/L) Fig. 4. Plots of the pH value versus the concentration of the hydrochloric acid in units of milli-mole per liter (mM/L) for the following three different types of waters: deionized water, tap water supplied from a municipal water supply system, and seawater. The square dots represent the acidity of the acidic water created from the deionized water, the circular dots represent the acidity of the acidic water made from the tap water, and the triangular dots represent the acidity of the acidic seawater.
log
NðtÞ N0
¼ 0:43anO3 s½1 exp ðt=sÞ;
(1)
where the constant N0 represents the initial density of the microorganisms, nO3 is the initial ozone density, and a is the inactivation coefficient of ozone in units of L/(mg s). The microbe number N in Eq. (1) in the unit volume can be estimated from Eq. (1) in terms of the time t. The curves in Fig. 5 were obtained from Eq. (1) for AOW, with nO3 ¼ 20 mg/L, s ¼ 8.1 s for pH ¼ 7, s ¼ 23 s for pH ¼ 5 and s ¼ 26 s for pH ¼ 4. These ozone decay times were measured values at an ethanol concentration of 0.072 mol/L. The parameter a ¼ 0.0215 L/(mg s) when obtaining the curves [13] here was the least-squares value fitted to the experimental data (triangular dots) for pH ¼ 7 in Fig. 5. Note that the ozone decay time s increases from s ¼ 8.1 s for pH ¼ 7 to s ¼ 23 s for pH ¼ 5 and to s ¼ 26 s for pH ¼ 4 in AOW at room temperature (25 C). The short decay time s ¼ 8.1 s in AOW at pH ¼ 7 is for a situation in which the environment contains many organic compounds, represented by an ethanol concentration of 0.072 mol/L corresponding to 3.4 g/L. The ozone concentration of 20 mg/L is far less than the ethanol concentration. However, the 20 mg/L ozone concentration is equivalent to 1.4 1017 molecules/cm3, which is much higher than the spore concentration on the order of 106/cm3. It is shown in Fig. 5 that the log of the ratio of
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H.S. Uhm et al. / Current Applied Physics 13 (2013) S30eS35
N to N0 for the acidic ozone water in the experimental data agrees remarkably well with the theoretical curves. The acidic ozone water may be also very effective for the inactivation of H1N1 viruses [16], also known as the swine flu virus, which appeared almost daily in the news during the summer of 2009 and which is a highly contagious disease. The sterilization of microbes in seawater [15] was carried out using ozone and acid in seawater. The spore concentration of the original spore suspension was 105e106 spores per mL. The sporetreatment experiments were conducted by adding 0.2 mL of the spore suspension to 10 mL of seawater at a specified pH value and at an ozone concentration of 5 mg/L. The concentration of ethanol in the mixture of 0.1 mL of the spore suspension and 10 mL of the seawater was 7.7 mM/L. Ozone in the water decayed rapidly at this ethanol concentration [15]. For example, the ozone decay time s in the seawater with an ethanol concentration of 7.7 mM/L was measured to be s ¼ 22 s for pH ¼ 8, s ¼ 40 s for pH ¼ 7, s ¼ 70 s for pH ¼ 6 and s ¼ 90 s for pH ¼ 5. One milliliter of the solution was obtained from each sample after a contact time of 40 min and was diluted with 9 mL of distilled water. The contact time t ¼ 40 min is much longer than the ozone decay time s of less than 2 min with a high concentration of ethanol. Eq. (1) is further simplified to [15]
log
NðtÞ ¼ 0:43anO3 s N0
(2)
for t >> s, which is typical for the sterilization of microbes in seawater. Fig. 6 shows the survival curve [15] for B. atrophaeus endospores exposed to seawater with a 5 mg/L ozone concentration at several different pH values. The horizontal scale represents the ozone decay time s measured in seconds corresponding to the specific pH value of seawater contaminated by an ethanol concentration of 7.7 mM/L. The vertical axis is the log of the ratio of the number of viable spores remaining (N) to the control number of N0. An untouched control was also analyzed each time to obtain the average control number N0 ¼ 3.3 105, which corresponds to log N0 ¼ 5.52. The dots in Fig. 6 represent the experimental data [15] pertaining to the log reduction of live microbes versus the ozone decay time s in seconds for seawater with an ozone concentration of 5 mg/L contaminated by an ethanol concentration of 7.7 mM/L corresponding to 360 mg/L. In effect, all of the spores were killed at
s ¼ 90 s, but one surviving spore at s ¼ 90 s was assumed for convenience regarding the log-scale plot shown in Fig. 6. The molecular number of ethanol in this seawater is 150 times greater than that of ozone. The straight line in Fig. 6 was obtained from Eq. (3) and was linearly fitted to the experimental dots (squares) with the parameter anO3 ¼ 0.135/s, which was the least-squares value fitted to the experimental data in Fig. 6. Assuming an initial ozone concentration of nO3 ¼ 5 mg/L, the inactivation coefficient [15] of ozone was calculated to be a ¼ 0.027 L/(mg s) for anO3 ¼ 0.135/s. The short decay time s in Fig. 6 represents a situation in which the seawater contains many organic compounds. On the other hand, for relatively clean seawater in an application to ballast water, the organic material is less than 5 mg/L and the ozone decay time at pH ¼ 7 is 3.3 min. Eq. (3) predicts the viable Bacillus spore number of N ¼ 8 for N0 ¼ 3.3 105 at an ozone concentration of nO3 ¼ 2 mg/L. Fig. 6 shows that the log of the ratio of N to N0 for the acidic seawater in the experimental data is in good agreement with the theoretical model. The ozone decay time of s ¼ 90 s at pH of 5 is four times that at a pH of 8. Therefore, an increase of the ozone decay time by lowering the pH value must play a pivotal role in the killing process. Similar sterilization can be achieved by a four-fold increase in the ozone concentration in seawater at pH ¼ 8. However, an ozone concentration of 20 mg/L in seawater may be impractical for sterilization applications. Hence, a reasonable ozone concentration at a low pH value may make it possible to sterilize a large amount of seawater in relatively little time, freeing this water from unwanted microbes. Fig. 6 clearly demonstrates that an increase of the ozone decay time at a low pH has the most important synergic effect on the sterilization of microbes in seawater. Other studies also show that acidic ozone water is a very effective means of sterilizing vegetative bacteria and viruses. Particularly, these studies indicated that acidic ozone water effectively disinfected enveloped and naked viruses in environments contaminated with numerous organic compounds. The acidic ozone water kills more than 99.98% of viruses, except for the CoxB3 virus [14]. Therefore, it is a good disinfectant of viruses. The HIV virus is more easily killed [14] than the FluA virus, although its death profile is similar to that of the FluA virus. Acidic ozone water may also be useful for disinfection in hospitals or other germ-infested areas. Finally, it is also concluded that acidic ozone water is applicable to the agricultural, seafood, and livestock industries to prevent the spoilage of products and to prolong the shelf lives of these products. 3. Discussion
Fig. 6. Plots of the survival curve for B. atrophaeus endospores exposed to seawater with a 5 mg/L ozone concentration at several different pH values. The horizontal scale represents the ozone decay time s measured in seconds corresponding to the specific pH value of seawater contaminated by an ethanol concentration of 7.7 mM/L.
According to the findings as given above, reactive oxygen species, particularly excited oxygen atoms and ozone molecules, are very effective for the sterilization of microbes. This leads to the question of what effects ROS (for example, acidic ozone water) has on advanced cells. To test this, an experiment was conducted on fertilized eggs. We injected tap water into 10 fertilized eggs, finding that all of these eggs were dead a few days after the water injection. It is likely that E. coli existing in the tap water may have caused the death of these eggs. We also injected acidic ozone water made of this tap water into 10 fertilized eggs. Remarkably, all of the eggs survived without any harm from the injection of the acidic ozone water. The E. coli typically existing in tap water was most likely eliminated by the acidic ozone water, which was then not harmful to the fertilized eggs. Thus, the ozone in the water may not be critically harmful to advanced living cells. A second experiment was related to the inactivation experiment involving the viruses. All viruses need a host to survive. Therefore, we used human cells or canine cells as the host cells for the viruses [14]. Therefore, we were forced to evaluate the survivability of the host cells in the stressful
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condition of the acidic ozone water, finding that all of the host cells survived well due to the treatment with acidic ozone water in a physical parameter range in which other microbes are destroyed. According to our previous observations of various microbes, the spore walls of B. atrophaeus (B. subtilis var. niger, ATCC 9372) are well coated with protein, whereas human or canine cells have membrane walls that are much more fragile than the Bacillus spore wall. Yet, canine cells are more resilient than Bacillus spore. Why are advanced cells such as canine cells more resilient than microbes in the acidic ozone water? We speculate that advanced cells have a variety of antioxidant enzymes, such as superoxide dismutase, which may protect the cells from the toxicity of the acidic ozone water. Hence, human cells may experience minimal damage from acidic ozone water at an intensity level that would otherwise exert a devastating effect on microbes. Reactive oxygen species including O*, O 2 , O3, NO, and H2O2 may have appeared when free oxygen became available in the earth’s atmosphere. The newly born cells in an oxygen-rich atmosphere may have developed a defense system against the toxicity of ROS. Enzymes such as superoxide dismutase may exist in these advanced cells, offering them protection against the threat of ROS toxicity. Meanwhile, these advanced cells may develop the capability of oxygen utilization by making use of certain enzymes, invigorating their activity and allowing this species to proliferate on earth. We would like to know what portion of DNA is responsible for the development of the antioxidant enzymes and what portion of DNA is responsible for the enzyme development as it pertains to oxygen utilization. Based on current evidence of volcanic activity, the first atmosphere on earth would have contained 60% hydrogen, 20% oxygen in the form of water vapor, 10% carbon dioxide, and 7% hydrogen sulfide. Major rainfall events led to the buildup of a vast ocean, enriching the other agents such as carbon dioxide, nitrogen and inert gases. The second atmosphere started about 3.4 billion years ago and mostly consisted of nitrogen. Hints of early life forms were found around this time period [17]. An oxygen-containing atmosphere began to develop about 1.7 billion years ago in the late Archaean eon, although the oxygen content was tenuous. Evidence of a third atmosphere can be seen with the development of red beds and the end of the banded iron formations, signifying a shift from a reducing atmosphere to an oxidizing atmosphere. Most free oxygen (O2) arose about 650 million years ago, fluctuating in its concentration, up and down, until it reached a steady state of more than 15 percent. Following this time span was the Phanerozoic eon, during which oxygen-breathing metazoan life forms began to appear. Meanwhile, simple cells such as prokaryotes appeared on earth around 3.8 billion years ago [18]. Complex cells in eukaryotes started to show up about 2 billion years ago. Multicellular life was initiated around 1 billion years ago. Advanced cells such as simple animals appeared on earth 600 million years ago, which coincides with the appearance of most of the free oxygen in the third atmosphere. Life on earth proliferated drastically about 600 million years ago due to the drastic increase in the oxygen content in the atmosphere, known as called Cambrian explosion. The accumulation of atmospheric oxygen allowed the formation of an ozone layer. This began to block ultraviolet radiation, permitting the colonization of the land. This suggests that advanced cells developed their capability to utilize oxygen by certain enzymes while protecting themselves from the toxicity of reactive oxygen species. 4. Conclusion A sterilization experiment with bacterial endospores indicates that an argoneoxygen plasma jet very effectively kills endospores
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of B. atrophaeus (ATCC 9372), thereby demonstrating its capability to clean surfaces and its usefulness for reinstating contaminated equipment as free from toxic biological agents. The key element of the sterilization process is the oxygen radicals. Transmission electron micrographs showed morphological changes in E. coli cells treated with atmospheric plasma at 75 W for 2 min. The treated cells showed severe cytoplasmic deformation and leakage of the bacterial chromosome material. The results clearly demonstrate the loss of viability of bacterial cells after the plasma treatment. Ozone is one of the most reactive oxygen species. Due to the synergic effects of the acidity and ozone, acidic ozone water is a very effective means of sterilizing various microbes. However, advanced cells such as human cells survive reasonably well after a treatment of acidic ozone water in the physical parameter range in which other microbes are devastated. This finding suggests that the enzymes such as superoxide dismutase may exist in these advanced cells, protecting them from the threat of ROS toxicity while these advanced cells develop the capability to utilize oxygen by making use of certain enzymes, invigorating their activity levels and allowing their species to proliferate on earth. This observation leads to many questions to be answered in future studies. The questions are as follow: How do antioxidant enzymes such as superoxide dismutase protect cells from the toxicity of ROS? Do other components of ROS show similar killing efficacy as ozone in acidic water? When do these enzymes appear in living cells on earth? Can we establish a timeline relationship between the evolution of living cells and the evolution of the atmosphere on earth? Can we find a relationship between ROS and the ROS defense system by making use of our ROS from plasma and ozone? We must design specific experimental procedures to answer all of these questions in future studies. Acknowledgements This work was financially supported by the SRC program (Grant #2010-0029421) of the National Research Foundation (NRF) and was also partially supported by the Research Grant of Kwangwoon University in 2013, and program funds from the Western Human Nutrition Research Center/ARS/USDA (USDA is an equal opportunity employer and provider). References [1] Y.C. Hong, H.S. Uhm, W.J. Yi, Appl. Phys. Lett. 93 (2008) 051504. [2] K.H. Becker, U. Kogelschatz, K.H. Schoenbach, R.J. Barker, Non-equilibrium Air Plasmas at Atmospheric Pressure, Institute of Physics, Bristol, 2005. [3] J. Park, I. Henins, H.W. Herrmann, G.S. Selwyn, R.F. Hicks, J. Appl. Phys. 89 (2001) 20. [4] M.A. Lieberman, A.J. Lichtenberg, Principles of Plasma Discharges and Materials Processing, second ed., Wiley, New York, 2005, pp. 270e279. [5] R.E. Walkup, K.L. Saenger, G.S. Selwyn, J. Chem. Phys. 84 (1986) 2668. [6] D. Pagnon, J. Amorim, J. Nahorny, M. Touzeou, M. Vialle, J. Phys. D 28 (1995) 1856. [7] S. Wang, V. Schulz-von der Garhen, H.F. Dobele, Appl. Phys. Lett. 83 (2003) 3272. [8] S.Z. Li, J.P. Lim, J.G. Kang, Han. S. Uhm, Phys. Plasmas 13 (2006) 093503. [9] J.P. Lim, H.S. Uhm, S.Z. Li, Appl. Phys. Lett. 90 (2007) 051504. [10] H.S. Uhm, J.P. Lim, S.Z. Li, Appl. Phys. Lett. 90 (2007) 261501. [11] J.P. Lim, H.S. Uhm, S.Z. Li, Phys. Plasmas 14 (2007) 093504. [12] Y.F. Hong, J.G. Kang, H.Y. Lee, H.S. Uhm, E. Moon, Y.H. Park, Lett. Appl. Microbiol. 48 (2009) 33. [13] H.Y. Lee, H.S. Uhm, Y.F. Hong, Y.H. Park, Appl. Phys. Lett. 92 (2008) 174102. [14] H.S. Uhm, H.Y. Lee, Y.C. Hong, D.H. Shin, Y.H. Park, Y.F. Hong, C.K. Lee, J. Appl. Phys. 102 (2007) 013303. [15] H.S. Uhm, Y.F. Hong, H.Y. Lee, Y.H. Park, J. Korean Phys. Soc. 56 (2010) 108. [16] H.S. Uhm, K.H. Lee, B.L. Seong, Appl. Phys. Lett. 95 (2009) 173704. [17] J. Schopf, Earth’s Earliest Biosphere: Its Origin and Evolution, Princeton University Press, Princeton, N.J, 1983. [18] Carl Woese, J. Peter Gogarten, When did eukaryotic cells (cells with nuclei and other internal organelles) first evolve? What do we know about how they evolved from earlier life-forms? Sci. Am. (October 21, 1999).