# Reactions of Microorganisms with Atomic Oxygen Radical Anions: Damage of Cells and Irreversible Inactivation

Authors: Longchun Li; Fan Wu; Yu Chen; Lei Xu; Xiaochang Hao; Yonghuan Chen; Yixiang Sun; Guoyin Xiong
Journal: Journal of Nanomaterials
 (2019)

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## Abstract

Reactive oxygen species play important effects on organisms not onlyin vivo but also in vitro. The atomic oxygen radical anion (O-) has shown extremely high oxidation and reactivity towards small molecules of hydrocarbons. However, the O- effects on cells of microorganisms are scarcely investigated. This work showed the evidence that O- could react quickly with microorganisms (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, Saccharomyces cerevisiae, and Actinomycetes (5046)) and damaged the cell walls seriously as well as their intrinsic structures, arising a fast and irreversible inactivation. SEM and TEM micrographs were used to reveal the structure changes of cells before and after reacting with O- radicals. The inactivation efficiencies of the microorganisms depended on the O- intensity, the initial population of microorganisms, the exposed area, the environment, and the microorganisms’ types. Over 99% reduction of an initial 1.0×107 colony-forming unit (cfu), E. coli population only required less than 2 minutes while exposed to a 0.23 μA/cm2 O- flux under dry argon atmosphere (30°C, 1 atm). The observation of anionic intermediates (CO-, CO2-, H2O-, and anionic hydrocarbons) by time-of-flight (TOF) mass spectrometry and the neutral volatile products (CO, CO2, and H2O) by quadrupole mass spectrometry (Q-MS) provided an evidence of the reactions of O- with hydrocarbon bonds of the microorganisms. The inactivation mechanism of microorganisms induced by O- was discussed.


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## Body

## 1. Introduction

Reactive oxygen species (ROS), such as the superoxide anion radical (O2-), the hydroxyl radical (OH), and the singlet oxygen (1O2), are key chemical species in the biochemical processes and exert a very important effect on organisms [1–6]. Organisms can suffer lipid peroxidation, protein denaturation, DNA injuries, and enzyme inactivation by ROS [7–13]. Due to high reactivity of ROS, all the cellar components, lipids, proteins, nucleic acids, and carbohydrates may be damaged by their reactions with ROS, giving rise to metabolic and cellular disturbances or cell death [14–17]. There are increasing evidences showing that ROS are responsible for many diseases such as inflammation, lung injury, ischaemia-reperfusion injury, cancer, and aging [18–20]. On the other hand, ROS are not always harmful to organisms. For instance, ROS may be used as the intracellular signaling species for the introduction of defense gene expression and destroying malignant cells or tissue [21, 22].Atomic oxygen radical anion (O-) is a monovalent anion (or monovalent negative ion) through the attachment of an electron to atomic oxygen (O). At the same time, O- is also considered as a radical because it has an unpaired electron in its outermost orbit. It was found that atomic O- had extremely high oxidation and reactivity towards the hydrocarbons’ small molecules [23–26]. Moreover, O- may be one of the most reactive oxygen species and therefore has various potential applications, such as a one-step synthesis of phenol from benzene [27], the reduction of NO [28], and the dissociation and oxidation of bio-oil [29, 30]. Previously, our work showed that the O- could quickly react with the B. subtilis cells and seriously damage the cell walls as well as their other contents, leading to a fast and irreversible inactivation [31].Herein, we investigated the O- effects on the microorganisms’ inactivation (including the B. subtilis cells), the changes of cell structures caused by O-, and the products formed by the reaction of the microorganisms with O-. Our results demonstrate that O- can quickly react with the microorganisms, including Gram-positive bacteria (B. subtilis, S. aureus), Gram-negative bacteria (E. coli, B. A. niger), and fungi (S. cerevisiae, Actinomycetes (5046)), and seriously damage the cell walls and their intrinsic structures, leading to a fast and irreversible inactivation. The observed intermediates and volatile products provided an important evidence of the reactions of O- with hydrocarbon bonds of the microorganisms.


## 2. Experimental Section

### 2.1. O- Source

A sustainable and high-purity O- source was developed by using the microporous crystal [Ca24Al28O64]4+·4O- as an O- emitter [32, 33]. We prepared the [Ca24Al28O64]4+·4O- crystal (C12A7-O-) by the solid-state reaction of CaCO3 and γ-Al2O3 (molar ratio of CaCO3:γ−Al2O3=12:7) under dry oxygen atmosphere. The powder mixtures were pressed to a pellet and sintered at 1350°C for 18 hours under flowing dried oxygen. The sintered product was cooled to 430°C slowly at a rate of ~10°C/min and then quickly quenched to room temperature. Recently, a special characteristic of C12A7-O-, emitting high intensity and purity O-, has been identified by our previous work [32, 33]. The emitted species from the C12A7-O- surface were about 90% O- and weak electron (<10%). A reflective field was used to reflect the electrons. We controlled the beam intensity of O- (0.01-2.0 μA/cm2) by changing the temperature of the C12A7-O- sample and the ion extraction field.


### 2.2. Microorganisms’ Preparation

The microorganisms investigated in our experiments were Gram-positive bacteria (B. subtilis, S. aureus), Gram-negative bacteria (E. coli, B. A. niger), and fungi (S. cerevisiae, Actinomycetes (5046)) which were purchased from China General Microbiological Culture Collection Center and maintained on nutrient agar slants at 5-6°C. Before the inactivation experiments, microorganisms were harvested by centrifuging (4000 rpm, 4°C) for 10 min and the microorganisms’ pellets were washed three times with sterile water. After pouring upper water, E. coli, B. subtilis, S. aureus, S. cerevisiae, and Actinomycetes (5046) were inoculated to L-broth (Bacto peptone 10 g/L, Bacto yeast extract 10 g/L, NaCl 5 g/L, pH=7.2) and then incubated at 37°C on a shaking tray for 12 hours. A. niger was cultured in potato dextrose broth (200 g peeled potato with a size of ~4.0×2.0×2.0cm3 were cooked in 1000 mL boiling distilled water for 8 min. After passing through four lay cheesecloths, the broth was collected and 20 g of dextrose was added.) and incubated at 27°C on a shaking tray for 12 hours. The density of microorganisms was controlled at 107~108 cfu/mL. Then, we transferred 0.1 mL resulted microorganisms to glass slide (20mm×20mm) for exposure to O- flux under the dry argon environment (1 atm). All petri dishes and glass microscopy slides used for holding the microorganisms were first autoclaved at 125°C for 1 h. The numbers of colonies on the slides before and after O- exposure were determined by the spread plate method with nutrient agar grown at 37°C for 12 h.E. coli spheroplasts were prepared by treating the intact E.coli cell suspension with 0.03 M Tris-HCl buffer (pH=8.1) containing 20% sucrose with lysozyme (1 mg/mL) and EDTA (ethylenediaminetetraacetate, 0.1 M pH=7.0) at 30°C for 60 min [34]. The treated cells were separated by centrifugation and resuspended in 0.2 M phosphate buffer (pH=6.6) containing 0.2% MgCl2 to form E. coli spheroplasts.


### 2.3. Inactivation Experiments

The experimental arrangement for measuring the inactivation efficiency and the reactions of microorganisms with O- was made up of three major parts: an O- radical source, a reaction chamber, and a product analysis system. The inactivation experiments were carried out in a cylindrical quartz reactor with a length of 60 cm and an inner diameter of 15 cm. The microorganism samples prepared were maintained by Petri dishes or slides, which were supported by a copper dish. The inactivation experiments can be performed under different environmental conditions by flowing different gases (such as Ar, He, N2, H2O, and O2). In order to study the atomic oxygen radical anion effects, all inactivation measurements were carried out in the dry argon environment. The reaction temperature was varied by cooling or heating copper dish with a water-cycled system. The O- flux emitted from the C12A7-O- sample was irradiated onto the microorganisms. The absolute O- ion flux was measured by a picoammeter and corrected by a TOF mass spectrometer. The numbers of colonies on the slides before and after O- exposure were determined by the spread plate method with nutrient agar grown at 37°C for 12 h.


### 2.4. Reaction Product Analysis

For online analysis of reaction products, a time-of-flight (TOF) mass spectrometry and a quadrupole mass (Q-MS) spectrometry were connected to the reactor. A time-of-flight (TOF) mass spectrometer was used for measuring the anionic intermediates formed by the reactions of microorganisms with O-. The neutral products were detected by a quadrupole mass (Q-MS) spectrometry.


### 2.5. SEM and TEM Measurements

SEM (scanning electron microscopy) and TEM (transmission electron microscopy) experiments were performed to study the structural changes of cells before and after exposure to O-. The preparation of test samples was similar to the inactivation experiments mentioned above. After exposure to O-, the suspensions of E. coli were fixed with an equal volume of 3% glutaraldehyde buffered at pH=7.2 with phosphate for about 1 h. Then, the samples were centrifuged, and the resulting bacterial pellets were exposed overnight to additional phosphate-buffered 3% glutaraldehyde solution. The glutaraldehyde-fixed bacteria were embedded in 1% agar and washed with phosphate buffer. After that, the samples were fixed in buffered 1% osmium tetroxide in cacodylate buffer for 1 h at room temperature and then dehydrated by successive soakings in 50, 70, 90, and 100% ethanol. The dried samples were rinsed twice with propylene oxide and infiltrated with propylene oxide-epoxy resin mixtures until the samples were in pure epoxy resin. Finally, the samples were placed in polyethylene capsules and resin polymerized at 60°C overnight [35]. Thin sections were cut using an ultracrotome and were mounted on 200 mesh copper grids and stained with uranyl acetate and lead citrate. The sections were examined with X-650 SEM (HITACHI) operating at 30 kV and H-800 TEM (HITACHI) operating at 100 kV.


## 2.1. O- Source

A sustainable and high-purity O- source was developed by using the microporous crystal [Ca24Al28O64]4+·4O- as an O- emitter [32, 33]. We prepared the [Ca24Al28O64]4+·4O- crystal (C12A7-O-) by the solid-state reaction of CaCO3 and γ-Al2O3 (molar ratio of CaCO3:γ−Al2O3=12:7) under dry oxygen atmosphere. The powder mixtures were pressed to a pellet and sintered at 1350°C for 18 hours under flowing dried oxygen. The sintered product was cooled to 430°C slowly at a rate of ~10°C/min and then quickly quenched to room temperature. Recently, a special characteristic of C12A7-O-, emitting high intensity and purity O-, has been identified by our previous work [32, 33]. The emitted species from the C12A7-O- surface were about 90% O- and weak electron (<10%). A reflective field was used to reflect the electrons. We controlled the beam intensity of O- (0.01-2.0 μA/cm2) by changing the temperature of the C12A7-O- sample and the ion extraction field.


## 2.2. Microorganisms’ Preparation

The microorganisms investigated in our experiments were Gram-positive bacteria (B. subtilis, S. aureus), Gram-negative bacteria (E. coli, B. A. niger), and fungi (S. cerevisiae, Actinomycetes (5046)) which were purchased from China General Microbiological Culture Collection Center and maintained on nutrient agar slants at 5-6°C. Before the inactivation experiments, microorganisms were harvested by centrifuging (4000 rpm, 4°C) for 10 min and the microorganisms’ pellets were washed three times with sterile water. After pouring upper water, E. coli, B. subtilis, S. aureus, S. cerevisiae, and Actinomycetes (5046) were inoculated to L-broth (Bacto peptone 10 g/L, Bacto yeast extract 10 g/L, NaCl 5 g/L, pH=7.2) and then incubated at 37°C on a shaking tray for 12 hours. A. niger was cultured in potato dextrose broth (200 g peeled potato with a size of ~4.0×2.0×2.0cm3 were cooked in 1000 mL boiling distilled water for 8 min. After passing through four lay cheesecloths, the broth was collected and 20 g of dextrose was added.) and incubated at 27°C on a shaking tray for 12 hours. The density of microorganisms was controlled at 107~108 cfu/mL. Then, we transferred 0.1 mL resulted microorganisms to glass slide (20mm×20mm) for exposure to O- flux under the dry argon environment (1 atm). All petri dishes and glass microscopy slides used for holding the microorganisms were first autoclaved at 125°C for 1 h. The numbers of colonies on the slides before and after O- exposure were determined by the spread plate method with nutrient agar grown at 37°C for 12 h.E. coli spheroplasts were prepared by treating the intact E.coli cell suspension with 0.03 M Tris-HCl buffer (pH=8.1) containing 20% sucrose with lysozyme (1 mg/mL) and EDTA (ethylenediaminetetraacetate, 0.1 M pH=7.0) at 30°C for 60 min [34]. The treated cells were separated by centrifugation and resuspended in 0.2 M phosphate buffer (pH=6.6) containing 0.2% MgCl2 to form E. coli spheroplasts.


## 2.3. Inactivation Experiments

The experimental arrangement for measuring the inactivation efficiency and the reactions of microorganisms with O- was made up of three major parts: an O- radical source, a reaction chamber, and a product analysis system. The inactivation experiments were carried out in a cylindrical quartz reactor with a length of 60 cm and an inner diameter of 15 cm. The microorganism samples prepared were maintained by Petri dishes or slides, which were supported by a copper dish. The inactivation experiments can be performed under different environmental conditions by flowing different gases (such as Ar, He, N2, H2O, and O2). In order to study the atomic oxygen radical anion effects, all inactivation measurements were carried out in the dry argon environment. The reaction temperature was varied by cooling or heating copper dish with a water-cycled system. The O- flux emitted from the C12A7-O- sample was irradiated onto the microorganisms. The absolute O- ion flux was measured by a picoammeter and corrected by a TOF mass spectrometer. The numbers of colonies on the slides before and after O- exposure were determined by the spread plate method with nutrient agar grown at 37°C for 12 h.


## 2.4. Reaction Product Analysis

For online analysis of reaction products, a time-of-flight (TOF) mass spectrometry and a quadrupole mass (Q-MS) spectrometry were connected to the reactor. A time-of-flight (TOF) mass spectrometer was used for measuring the anionic intermediates formed by the reactions of microorganisms with O-. The neutral products were detected by a quadrupole mass (Q-MS) spectrometry.


## 2.5. SEM and TEM Measurements

SEM (scanning electron microscopy) and TEM (transmission electron microscopy) experiments were performed to study the structural changes of cells before and after exposure to O-. The preparation of test samples was similar to the inactivation experiments mentioned above. After exposure to O-, the suspensions of E. coli were fixed with an equal volume of 3% glutaraldehyde buffered at pH=7.2 with phosphate for about 1 h. Then, the samples were centrifuged, and the resulting bacterial pellets were exposed overnight to additional phosphate-buffered 3% glutaraldehyde solution. The glutaraldehyde-fixed bacteria were embedded in 1% agar and washed with phosphate buffer. After that, the samples were fixed in buffered 1% osmium tetroxide in cacodylate buffer for 1 h at room temperature and then dehydrated by successive soakings in 50, 70, 90, and 100% ethanol. The dried samples were rinsed twice with propylene oxide and infiltrated with propylene oxide-epoxy resin mixtures until the samples were in pure epoxy resin. Finally, the samples were placed in polyethylene capsules and resin polymerized at 60°C overnight [35]. Thin sections were cut using an ultracrotome and were mounted on 200 mesh copper grids and stained with uranyl acetate and lead citrate. The sections were examined with X-650 SEM (HITACHI) operating at 30 kV and H-800 TEM (HITACHI) operating at 100 kV.


## 3. Results and Discussion

### 3.1. Destruction of Cell Structures Caused by O-

SEM and TEM micrographs were used to disclose any changes in the cell structures (the wall, the membrane, or the nucleoid) of the microorganisms induced by O-. Figures 1(a) and 1(b) present the SEM photographs for E. coli cells before (Figure 1(a)) and after the O- exposure (Figure 1(b)). The irradiated cells appeared dramatically swollen and collapsed with a large amount of fragments, which were associated with the damages to the cell walls and subsequent lysis due to the O- irradiation. Figures 1(c) and 1(d) show the representative TEM micrographs of the initial (Figure 1(c)) and the O--irradiated (Figure 1(d)) E. coli cells, respectively. The lighter part of the cell was the nuclear region containing some DNA fibrils and electron-dense ribosomes. The nucleoids after the exposure had contracted and a coarse precipitation of DNA appeared (Figure 1(d)), which indicated that O- might be able to penetrate the cells, and reacted with proteins or numerous enzymes involved in the control of DNA conformation in the nucleoids, resulting in the precipitation of DNA. Some of the cells appeared to be disrupted, and fragments of lysed cells were observed. The TEM results reveal that the secondary structure of the DNA-binding proteins of the cells was destroyed by the exposure to O-. These results are in good agreement with our earlier research [31].Figure 1
(a) SEM image of the initialE. coli cells (image size: 13.7×11.4cm2, initial number: N0=1.0×108cfu/mL). (b) SEM image of the exposed E. coli cells (O- flux intensity: 192 nA/cm2, exposure time: 10 min, and reaction temperature: 30°C). (c) TEM image of the initial E. coli cells (image size: 7.4×5.1cm2, N0=5×107cfu/mL). (d) TEM image of the exposed E. coli cells (O- flux intensity: 135 nA/cm2, exposure time: 10 min, and reaction temperature: 30°C).
(a)
(b)
(c)
(d)


### 3.2. O- Effects on the Microorganisms’ Inactivation

The correlation between the inactivation efficiency of the microorganisms and the O- intensity was also demonstrated by measuring the survival curves under different O- intensity. Figure 2(a) displays a series of survival diagrams under different O- intensity for E. coli intact cells, which exhibits biphasic curves (fast and slow processes). The decay of the survival numbers of E. coli cells, both in the fast and slow phases, depends on the O- intensity. On the other hand, the survival curves of E. coli spheroplasts in the fast processes are much different from those of E. coli intact cells (Figure 2(b)). The survival numbers of E. coli spheroplasts quickly decreased to the slow phases within 1 min while E. coli intact cells require about 5 min at the same condition. This result indicates that O- destroy cell walls firstly, which agree with the result by the SEM measurements.Figure 2
(a) Survival curves ofE. coli cells (N0=1.0×107cfu/mL) exposed to a series of O- flux intensity (exposure size: 1.8 cm2). (b) Survival curves of E. coli spheroplasts (N0=1.0×107cfu/mL) exposed to a series of O- flux intensity (exposed size: 1.8 cm2), reaction temperature: 30°C.
(a)
(b)For the biphasic inactivation processes, we deduce that during the first phase; the O- radicals react with the cell walls and form the volatile small molecules (such as CO, CO2, and H2O), resulting in quickly irreversible damage and lysis of the cells in the top layer (Figure 1(b)). As the microorganisms are killed in the first phase, the inactivated cells may stack and form an isolated layer. The occurrence of slow processes may be attributed to protection resulting from the contents of dead cells, which shielded the remaining survivors or aggregation during the treatment. The second phase, therefore, would mainly reflect the time required for sufficient erosion by reacting with the debris on the top layer.O- effects on the other microorganisms’ inactivation were also observed. As shown in Figure 3, the O--induced inactivation efficiencies for B. subtilis, S. aureus, A. niger, S. cerevisiae, and Actinomycetes (5046) were also investigated. We found Actinomycetes (5046) were most resistant to the O- interaction, because Actinomycetes (5046) are terrestrial prokaryotes that grow mainly in mycelia and reproduce by spore. On the other hand, fungi (A. niger, S. cerevisiae) were easiest to inactivate in the same O- intensity. It appears that Gram-negative bacteria (E. coli) are more sensitive to O- than Gram-positive bacteria (B. subtilis, S. aureus). Thus, the inactivation efficiencies depend on the microorganisms’ types, because the microorganisms with different components may have different resistant ability to O-.Figure 3
Survival curves of different types of microorganisms exposed to O-: B. subtilis, S. aureus, A. niger, E. coli, S. cerevisiae, and Actinomycetes (5046). N0: 1.0×107cfu/mL, O- flux intensity: 52 nA/cm2, reaction temperature: 30°C, exposed size: 1.8 cm2.Moreover, the inactivation efficiencies also depend on the reaction temperature and the exposure area. Figure4 presents the survival curves of E. coli at different reaction temperature (29°C, 34°C, and 42°C). With elevatory reaction temperature, the inactivation efficiency of E. coli increases, which indicate that the inactivation process of E. coli by O- is a thermally enhanced process. In addition, with enlarged exposure area, the inactivation efficiency also increases.Figure 4
Reaction temperature effect on survival curves of E. coli exposed to O-, N0: 1.0×107cfu/mL, O- flux intensity: 52 nA/cm2, exposure size: 1.8 cm2.


### 3.3. Products Formed by the Reaction ofE. coli with O-

The reaction products had been detected by the online analysis of the trail gases when O- is exposed onto the microorganisms. The anionic intermediates and the neutral products were detected by a time-of-flight spectrometry (TOF) and a quadrupole mass (Q-MS) spectrometry, respectively. Figure 5(a) displays a typical TOF spectrum for the O-/E. coli reaction system. The anionic species of H-, OH-, CO-, CO2-, H2O-, and anionic hydrocarbons were clearly identified (Figure 5(a)). In addition, the neutral products of CO, CO2, and H2O were also observed by the Q-MS spectra (Figure 6(a)). It was also found that the peak intensities both for the anionic species and the neutral products increased with the increasing of the O- flux intensity (Figure 5(b) and Figure 6(b)). The microorganisms, briefly, are macromolecules mainly consisted of carbon, hydrogen, oxygen, and nitrogen atoms. Among these elements, carbon atom is the only one not self-associating to make volatile molecules such as CO and CO2, and it must therefore form volatile compounds with other atoms in order to be removed from the cell surface. The released CO and CO2 would originate from the reactions of O- with hydrocarbon bonds of the microorganisms’ cells. Based on the observations mentioned above, the active O- radical anions can react with the microorganisms, damage their cell structures, and finally result in a fast and irreversible inactivation. The inactivation mechanism of microorganisms induced by O- was schematically described in Figure 7.Figure 5
(a) Typical TOF spectrum shows the anionic intermediate species formed in the O--induced inactivation processes (O-: 250 nA/cm2). (b) The intensity changes of H2O-, CO-, and CO2- with different O- intensity. Sample: E. coli, N0: 1.0×1010cfu/mL, exposure size: 1.8 cm2.
(a)
(b)Figure 6
(a) Typical Q-MS spectrum shows that the volatile small molecules of CO, CO2, and H2O were formed in the O--induced inactivation processes (O-: 250 nA/cm2). (b) The intensity changes of H2O, CO, and CO2 with different O- intensity. Sample: E. coli, N0: 1.0×1010cfu/mL, exposure size: 1.8 cm2.
(a)
(b)Figure 7
A schematic diagram shows the inactivation process induced by the O- reactions with the macromolecules of the outer wall and the inner content of the microorganism cell.


## 3.1. Destruction of Cell Structures Caused by O-

SEM and TEM micrographs were used to disclose any changes in the cell structures (the wall, the membrane, or the nucleoid) of the microorganisms induced by O-. Figures 1(a) and 1(b) present the SEM photographs for E. coli cells before (Figure 1(a)) and after the O- exposure (Figure 1(b)). The irradiated cells appeared dramatically swollen and collapsed with a large amount of fragments, which were associated with the damages to the cell walls and subsequent lysis due to the O- irradiation. Figures 1(c) and 1(d) show the representative TEM micrographs of the initial (Figure 1(c)) and the O--irradiated (Figure 1(d)) E. coli cells, respectively. The lighter part of the cell was the nuclear region containing some DNA fibrils and electron-dense ribosomes. The nucleoids after the exposure had contracted and a coarse precipitation of DNA appeared (Figure 1(d)), which indicated that O- might be able to penetrate the cells, and reacted with proteins or numerous enzymes involved in the control of DNA conformation in the nucleoids, resulting in the precipitation of DNA. Some of the cells appeared to be disrupted, and fragments of lysed cells were observed. The TEM results reveal that the secondary structure of the DNA-binding proteins of the cells was destroyed by the exposure to O-. These results are in good agreement with our earlier research [31].Figure 1
(a) SEM image of the initialE. coli cells (image size: 13.7×11.4cm2, initial number: N0=1.0×108cfu/mL). (b) SEM image of the exposed E. coli cells (O- flux intensity: 192 nA/cm2, exposure time: 10 min, and reaction temperature: 30°C). (c) TEM image of the initial E. coli cells (image size: 7.4×5.1cm2, N0=5×107cfu/mL). (d) TEM image of the exposed E. coli cells (O- flux intensity: 135 nA/cm2, exposure time: 10 min, and reaction temperature: 30°C).
(a)
(b)
(c)
(d)


## 3.2. O- Effects on the Microorganisms’ Inactivation

The correlation between the inactivation efficiency of the microorganisms and the O- intensity was also demonstrated by measuring the survival curves under different O- intensity. Figure 2(a) displays a series of survival diagrams under different O- intensity for E. coli intact cells, which exhibits biphasic curves (fast and slow processes). The decay of the survival numbers of E. coli cells, both in the fast and slow phases, depends on the O- intensity. On the other hand, the survival curves of E. coli spheroplasts in the fast processes are much different from those of E. coli intact cells (Figure 2(b)). The survival numbers of E. coli spheroplasts quickly decreased to the slow phases within 1 min while E. coli intact cells require about 5 min at the same condition. This result indicates that O- destroy cell walls firstly, which agree with the result by the SEM measurements.Figure 2
(a) Survival curves ofE. coli cells (N0=1.0×107cfu/mL) exposed to a series of O- flux intensity (exposure size: 1.8 cm2). (b) Survival curves of E. coli spheroplasts (N0=1.0×107cfu/mL) exposed to a series of O- flux intensity (exposed size: 1.8 cm2), reaction temperature: 30°C.
(a)
(b)For the biphasic inactivation processes, we deduce that during the first phase; the O- radicals react with the cell walls and form the volatile small molecules (such as CO, CO2, and H2O), resulting in quickly irreversible damage and lysis of the cells in the top layer (Figure 1(b)). As the microorganisms are killed in the first phase, the inactivated cells may stack and form an isolated layer. The occurrence of slow processes may be attributed to protection resulting from the contents of dead cells, which shielded the remaining survivors or aggregation during the treatment. The second phase, therefore, would mainly reflect the time required for sufficient erosion by reacting with the debris on the top layer.O- effects on the other microorganisms’ inactivation were also observed. As shown in Figure 3, the O--induced inactivation efficiencies for B. subtilis, S. aureus, A. niger, S. cerevisiae, and Actinomycetes (5046) were also investigated. We found Actinomycetes (5046) were most resistant to the O- interaction, because Actinomycetes (5046) are terrestrial prokaryotes that grow mainly in mycelia and reproduce by spore. On the other hand, fungi (A. niger, S. cerevisiae) were easiest to inactivate in the same O- intensity. It appears that Gram-negative bacteria (E. coli) are more sensitive to O- than Gram-positive bacteria (B. subtilis, S. aureus). Thus, the inactivation efficiencies depend on the microorganisms’ types, because the microorganisms with different components may have different resistant ability to O-.Figure 3
Survival curves of different types of microorganisms exposed to O-: B. subtilis, S. aureus, A. niger, E. coli, S. cerevisiae, and Actinomycetes (5046). N0: 1.0×107cfu/mL, O- flux intensity: 52 nA/cm2, reaction temperature: 30°C, exposed size: 1.8 cm2.Moreover, the inactivation efficiencies also depend on the reaction temperature and the exposure area. Figure4 presents the survival curves of E. coli at different reaction temperature (29°C, 34°C, and 42°C). With elevatory reaction temperature, the inactivation efficiency of E. coli increases, which indicate that the inactivation process of E. coli by O- is a thermally enhanced process. In addition, with enlarged exposure area, the inactivation efficiency also increases.Figure 4
Reaction temperature effect on survival curves of E. coli exposed to O-, N0: 1.0×107cfu/mL, O- flux intensity: 52 nA/cm2, exposure size: 1.8 cm2.


## 3.3. Products Formed by the Reaction ofE. coli with O-

The reaction products had been detected by the online analysis of the trail gases when O- is exposed onto the microorganisms. The anionic intermediates and the neutral products were detected by a time-of-flight spectrometry (TOF) and a quadrupole mass (Q-MS) spectrometry, respectively. Figure 5(a) displays a typical TOF spectrum for the O-/E. coli reaction system. The anionic species of H-, OH-, CO-, CO2-, H2O-, and anionic hydrocarbons were clearly identified (Figure 5(a)). In addition, the neutral products of CO, CO2, and H2O were also observed by the Q-MS spectra (Figure 6(a)). It was also found that the peak intensities both for the anionic species and the neutral products increased with the increasing of the O- flux intensity (Figure 5(b) and Figure 6(b)). The microorganisms, briefly, are macromolecules mainly consisted of carbon, hydrogen, oxygen, and nitrogen atoms. Among these elements, carbon atom is the only one not self-associating to make volatile molecules such as CO and CO2, and it must therefore form volatile compounds with other atoms in order to be removed from the cell surface. The released CO and CO2 would originate from the reactions of O- with hydrocarbon bonds of the microorganisms’ cells. Based on the observations mentioned above, the active O- radical anions can react with the microorganisms, damage their cell structures, and finally result in a fast and irreversible inactivation. The inactivation mechanism of microorganisms induced by O- was schematically described in Figure 7.Figure 5
(a) Typical TOF spectrum shows the anionic intermediate species formed in the O--induced inactivation processes (O-: 250 nA/cm2). (b) The intensity changes of H2O-, CO-, and CO2- with different O- intensity. Sample: E. coli, N0: 1.0×1010cfu/mL, exposure size: 1.8 cm2.
(a)
(b)Figure 6
(a) Typical Q-MS spectrum shows that the volatile small molecules of CO, CO2, and H2O were formed in the O--induced inactivation processes (O-: 250 nA/cm2). (b) The intensity changes of H2O, CO, and CO2 with different O- intensity. Sample: E. coli, N0: 1.0×1010cfu/mL, exposure size: 1.8 cm2.
(a)
(b)Figure 7
A schematic diagram shows the inactivation process induced by the O- reactions with the macromolecules of the outer wall and the inner content of the microorganism cell.


## 4. Conclusions

The active O- radical anions possess high reactivity towards the microorganisms and damage the cell walls as well as their intrinsic structures, resulting in a fast and irreversible inactivation. The inactivation efficiencies depend on the O- flux intensity, the initial population of microorganisms, the exposed area, environment, as well as the microorganisms’ types. The observation of anionic intermediates (CO-, CO2-, H2O-, and anionic hydrocarbons) by time-of-flight (TOF) mass spectrometry and the neutral volatile products (CO, CO2, and H2O) by quadrupole mass spectrometry (Q-MS) provided an evidence of the reactions of O- with hydrocarbon bonds of the microorganisms. We believe that the present approach would be extended to other fields such as the O- effects on lipid, protein, DNA, enzyme, and virus.



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*Source: 2483060-2019-11-03.xml*