Rapidly eliminating pathogenic microorganisms in large air space using spraying •OH radicals

Abstract

A new method for rapidly eliminating pathogenic microorganisms in large air space using spraying •OH radicals is presented in this paper. With a physical method of strong electric-field discharge, large numbers of •OH radicals were produced by the oxygen activated particles of O₂ ⁺, O(¹D), O(³P), etc., and the introducing reagent HO₂ ⁻. The gram-positive bacteria Bacillus subtilis, the gram-negative bacteria Serratia marcescens, and Bacillus spores were used for the eliminating experiments. Results show that the different microorganisms were rapidly killed by •OH radicals with a concentration of 0.8 mg/L and spraying density of 21 μL/m² within 4 sec. Cell morphological changes were also observed under microscope. The cells of B. subtilis and Bacillus spores in their cellular wall, cellular membrane, or cell protoplasm were greatly destroyed when being exposed to a killing dosage of •OH radicals.

Implications:

Increasing air spread of pathogenic microorganisms has prompted serious concerns for their threat to environmental safety. However, there is no an effective method to rapidly eliminate these harmful microorganisms in large air space. In comparison with conventional disinfectants, advanced oxidation technology (AOT) based on hydroxyl radicals (•OH) has excellent advantages. The main problem of how to produce the •OH radicals with high concentration and large production is solved by a physical method of strong electric-field discharge. A new method for rapidly eliminating pathogenic microorganisms in large air space using spraying •OH radicals is described in this paper.

Introduction

As the environmental pollution is getting worse nowadays, pathogenic microorganisms suspended in air space are extraordinary active, resulting in a higher incidence of infectious diseases than ever before, and are extremely threatening human health and the survival of animals and plants. Between the months of November 2002 and July 2003, severe acute respiratory syndrome (SARS) rapidly spread from China to other 37 countries around the world, leading to 775 human deaths with an economic loss of 40 billion US dollars (Smith, 2006). In early 2009, a new strain of swine-origin H1N1 spread worldwide from Mexico. This novel H1N1 was declared as an influenza pandemic by the World Health Organization (WHO) (World Health Organization, 2010), causing about 17,000 human deaths by the start of 2010. Therefore, it is very important to develop a rapid and efficient method for the elimination of pathogenic microorganisms in large air space.

Currently, chlorine, alkali, and alkali-alcolol-amine as the three kinds of main chemical disinfectants are widely used for eliminating microbial contamination. However, some drawbacks (Räber and McGuire, 2002; Raber et al., 2001) are as follows: (1) With the selectivity, a kind of chemical disinfectant could only correspondingly kill one or several kinds of pathogenic microorganisms. (2) The processing time is long, in the range of 0.5∼1 hr owing to the low chemical reaction rate. (3) The lethal dosage value is very high, which could reach 9% (v/v), resulting in a serious secondary pollution by the remaining chlorine compounds. (4) The lethal processing is limited on the surface of objects, impossible to be applied to large air space.

In comparison with the above chemical disinfectants, advanced oxidation technology (Glaze et al., 1987; Roberto and Vincenzo, 1999) (AOT)-based on hydroxyl radicals (•OH) has several excellent advantages: (1) Without selectivity, the •OH radicals can kill any pathogenic microorganisms in low lethal dosage because of its strong oxidizability whose oxidation potential is 2.8 V, only less than that of fluorine. (2) The •OH processing time is very short, within several seconds. The reason is that chemical reaction rate of •OH is up to 10⁹ L·mol⁻¹·sec⁻¹, which is 10⁷ times higher than that of other oxidants such as O₃, H₂O₂, Cl₂, and so on. (3) As a green oxidant, the •OH radicals are decomposed into H₂O and O₂ without any residual oxidant after its biochemical reactions.

The main problem for rapidly eliminating pathogenic microorganisms in large air space is how to produce the •OH radicals with high concentration and large production. Currently, the main methods are Fenton catalysis (Shreya et al., 2007; Veressinina and Trapido, 2008), photocatalysis (Maurizio and Vincenzo, 2005), ozone (Naoyuki and Yukako, 2005), water injection (Nick and Satoshi, 2010), as well as their collaborative effects. However, these technologies have some serious disadvantages: (1) The •OH radicals are obtained in a low concentration and low amount of production, so that the whole biochemical reaction time is long, in the range of 15∼360 min. The above-mentioned technologies are only applied to the small-scale experiments or applications. (2) Large numbers of medicaments or catalysts such as H₂O₂, TiO₂, Fe²⁺, and so on are necessary in the process of •OH production, resulting in its high cost as well as the safety problem of H₂O₂. (3) In order to increase the •OH production, several kinds of technologies are collaborated together resulting in large-volume accessory equipments such as the bubble tower, rotating packed bed, and so on.

In our previous studies, large number of •OH radicals were produced by the ionization and dissociation of O₂ in air and H₂O at gas state using a physical method of strong electric-field discharge. And the •OH radicals have been successfully used in the treatment of ship's ballast water and red-tide in ocean (Bai et al., 2005a, 2005b). In this paper, the killing effects of •OH radicals on the pathogenic microorganisms and their cell morphological changes are presented.

Formation of •OH Radicals

In order to obtain larger number of •OH radicals, the electron energy has to be higher than the ionization potential of O₂ and H₂O (12.5 and 12.6 eV, respectively) and dissociation potential of O₂ (8.4 eV), respectively. With a method of strong electric-field discharge (SEFD), the electrons have the average energy of 9 eV, accounting for a lot of electrons to have enough high energy to ionize and dissociate O₂ and H₂O.

The plasma processes of •OH formation are shown in Figure 1, parts (a) to (c).

Figure 1. Plasma chemistry processes of •OH formation.

In part (a), the O₂ molecules are ionized and dissociated into the oxygen active particles of O₂ ⁺, O(¹D), O(³P), etc. With a very short life time, the O(¹D) and O(³P) rapidly react with O₂ to form ozone (O₃). The concentration of O active particles reaches 240 mg/L (Bai et al., 2000b), 3 times higher than that of current plasma techniques of about 70 mg/L (Bai et al., 2000a).

In part (b), the H₂O molecules at a gas state are ionized and excited into H₂O⁺ and H₂O* to further form H₂O₂. The content of H₂O₂ is about 0.34 μg/L. The H₂O₂ loses an H⁺ to form HO₂ ⁻, a key introducing reagent of •OH, by an equilibrium reaction. HO₂ ⁻ reaction rate constant is 2.2 × 10⁶L·mol⁻¹·sec⁻¹, which is 30 thousand times higher than that of HO⁻ introducing reagent with only 70 L·mol⁻¹·sec⁻¹ (Xu and Zhao, 2003).

In part (c), the •OH radicals are generated by the three plasma processes (1), (2), and (3). When the electron energy is between 8.4 and 12.5 eV, the •OH radicals are formed mainly from the O₃ with HO₂ ⁻ following the reaction of (1) (Glaze et al., 1987; Staehelin and Hoigne, 1982). When the electron energy is higher than 12.5 eV, the •OH radicals come mainly from O₂ ⁺ with H₂O to form water cluster ions O₂ ⁺ (H₂O) and then the dissociative reactions following the reaction of (2) (Penetrante et al., 1997). In addition, the •OH radicals are produced from H₂O⁺ reacting with water cluster electrons e⁻ _aq following the reaction of (3).

The •OH radicals in water involve very complicating chain reactions, which are very quickly to convert into other active particles such as O₂ ^−•, O₃ ^−•, HO₃ ^•, HO₂ ⁻, HO₂ ^•, and so on, meanwhile rapidly killing the pathogenic microorganisms.

Experiment

Experimental system

The experimental system for killing pathogenic microorganisms in air space is shown in Figure 2. The O₂ enriched from air with a purity of 98.5% and the H₂O at gas state (3.5%, v/v) were introduced into the plasma reactor. With the strong electric-field discharge (SEFD), the active particles of O₂ ⁺, O₃, and H₂O⁺, H₂O₂ were produced to together inject into a gas/liquid dissolver 4. In water pipe with the high pressure injecting of dissolver 4, the •OH radicals and other oxygen active particles were formed to further dissolve into water through the gas/liquid separator 5. The remaining gases were removed by electric heating with eliminator 6. With a nozzle, large numbers of •OH fogdrops were sprayed to the body with microorganisms by BP-1035 electric sprayer. The treating capacity of the equipment is 0.5 m³/hr.

Figure 2. The system for killing pathogenic microorganisms in air space: 1, 11 = valves; 2 = liquid flowmeter; 3 = pump; 4 = gas/liquid dissolver; 5 = gas/liquid separator; 6 = eliminator of residual gas; 7 = fogdrops; 8 = body with microorganisms; 9 = controller; 10 = transformer; 12 = gas flowmeter; 13 = plasma reactor; 14 = check valve.

The closing configuration of plasma reactor is shown in Figure 3, with the dimensions of 230 (length) × 150 (width) × 40 (height) mm³. The strong electric-field discharge plasma was filled in two microgaps (width = 0.47 mm) at an atmospheric pressure and ambient temperature. Two α-Al₂O₃ dielectric layers, with the thickness of 0.2 mm, dielectric constant of 10, and the insulation intensity of 350 kV/cm, were covered on the discharge electrode of Ag thin plate (180 × 90 mm²). The self-made power supply was applied on the discharge electrodes with the following parameters: applied voltage, 2.5 kV; frequency, 10.4 kHz; current pulse width, 5∼10 ns. The electric bridge techniques of high voltage are used to measure the discharge power (Zhang et al., 2003), which can efficiently eliminate the measure error from the capacitor of discharge gap.

Figure 3. Configuration of plasma reactor.

Test of •OH radicals

As for the measurement of •OH concentration, ortho-hydroxybenzoic acid (salicylic acid, SA) was employed as a probe reagent, and the method was described previously (Jen et al., 1998; Quan et al., 2007). When the SA was applied in the gas/liquid dissolver, the rapid reaction between SA and •OH radicals resulted in the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Then the •OH concentration was obtained by testing the production of these two compounds through high-performance liquid chromatography (HPLC; Dionex, Sunnyvale, CA, USA). As shown in Figure 4, the peaks of 2,3-DHBA and 2,5-DHBA are obviously identified in the liquid chromatogram of SA hydroxyl compounds, indicating that •OH radicals assuredly existed in water. The concentration of other oxygen active particles was converted into the •OH concentration according to their oxidation potential.

Figure 4. Liquid chromatogram of SA hydroxyl compounds.

The spray density of •OH radicals was calculated from the flow rate of •OH solution, spray time, and spray area as shown in eq 1. The flow rate and the spray area were constants in this experimental system.

(1)

Testing methods

The gram-positive bacteria Bacillus subtilis, gram-negative bacteria Serratia marcescens, and Bacillus spores were used for the killing experiments of microorganisms. Cells were inoculated with a concentration of 10⁷ colony-forming units (CFU)/mL in liquid cultures. Then 0.1 mL cell suspension was transferred onto Petri dish (diameter 60 mm) and distributed evenly in the center. The electric sprayer was placed vertically above the Petri dishes, working with different spray time to get various spray density of •OH radicals. After •OH radical treatment, sterile neutralizer (containing 5 g/L Na₂S₂O₃ and 0.1% peptone) and dilution were added into the Petri dish immediately. The neutralizer was used to neutralize the redundant •OH radicals and other oxygen active particles for controlling the proceeding time. The survival numbers (CFU) of cells were determined by standard plate count method after incubated at 37 °C for 48 hr. Each experiment had three samples for each microorganism. The morphological changes of the microorganisms were also observed under microscope after •OH treatment.

Experimental Results

Effect of •OH concentration on killing efficiency

The effect of •OH concentration on killing efficiency of microorganisms is shown in Figure 5 under the conditions of spraying density of 21 μL/cm² and processing time of 4 sec. For S. marcescens, the survival cells decreased dramatically when the •OH concentration was above 0.15 mg/L. There was nearly no survival cells when •OH concentration was 0.41 mg/L. Similar results were obtained for B. subtilis cells. The survival B. subtilis cells decreased to undetectable levels when the •OH concentration reached 0.5 mg/L. However, the results are different when Bacillus spores were treated. The survival cell numbers of Bacillus spores dropped significantly when the •OH concentration was greater than 0.3 mg/L, and cells became undetectable when the concentration was increased to 0.8 mg/L. In order to kill all kinds of microorganisms, the •OH concentration of 0.8 mg/L was applied to the following experiments.

Figure 5. •OH concentration and the survival cell numbers (CFU) of microorganisms.

Effect of •OH spraying density on killing efficiency

The effect of •OH spraying density on killing efficiency is shown in Figure 6. When the •OH concentration was 0.8 mg/L with the processing time of 4 sec on the infected plate, the CFU numbers of all the cells were decreased very quickly with the increase of •OH spray density from 0 to about 21 μL/cm². There are three similar curves in Figure 6, the lethal spraying density of •OH radicals for S. marcescens is 19 μL/cm², and for B. subtilis and Bacillus spores are 21 μL/cm².

Figure 6. •OH spray density and the survival cell numbers (CFU) of microorganisms.

Effect of •OH processing time on killing efficiency

Since the wide spreading of pathogenic microorganisms is usually very fast, it is important to control the spread within a short time. Therefore, the experiment about the effect of •OH processing time on kill efficiency was carried out, with the •OH concentration of 0.8 mg/L and spray density of 21 μL/cm². The relationship between processing time and survival cell numbers is shown in Figure 7. One second after •OH treatment, the CFU numbers of B. subtilis decreased dramatically from 10⁵ to about 100, and that of S. marcescens decreased from 10⁵ to about 200. There were no survival cells for them after 3 sec. For Bacillus spores, the CFU numbers quickly dropped down 1 sec after •OH treatment, and then turned to 0 after 4 sec. Although the processing time of bacteria and spores are different, the killing time is very short, just in few seconds. The experimental results indicate that it is a rapid and efficient approach for eliminating pathogenic microorganisms in large air space by spraying •OH radicals.

Figure 7. •OH processing time and the survival cell numbers (CFU) of microorganisms.

Morphological changes of microorganisms

The morphological changes of B. subtilis and Bacillus spores with the •OH treatment were also observed under microscope. As shown in Figure 8, before •OH treatment, the rod-shaped B. subtilis were clearly observed that cells were intact and healthy and the cytoplasm was evenly distributed. After •OH treatment, the membrane integrity was largely lost and their cytoplasm was distributed in clumps (Figure 8a). Compared with B. subtilis, the cell of Bacillus spores with a tough outer coating consists of several layers, such as exosporium, spore coat, cortex, core wall, and so on, so that it has very strong capacity to resist the destruction of bad living conditions (Heninger et al., 2009). Therefore, Bacillus spores is impossible to be rapidly killed by conventional chemical disinfectants such as chlorine, alkali, and alkali-alcolol-amine. However, after •OH treatment, the Bacillus spores were also disrupted, lysed into debris, and the round-shaped cells disappeared (Figure 8b).

Figure 8. Cell micrographs (10 × 40) of Bacillus subtilis (a) and Bacillus spores (b) before and after •OH treatment.

Because of the different cell structure, Bacillus spores are more difficult to be killed than B. subtilis and S. marcescens. Correspondingly, the Bacillus spores need higher •OH killing concentration, spray density, and time. The concentration, spray density, and processing time are the three important parameters for the killing effect of •OH radicals on microorganisms. Treating microorganisms with different cell structures, different concentration, spray density, and processing time of •OH radicals are needed.

Conclusions

A new method was successfully investigated for rapidly eliminating the pathogenic microorganisms in large air space using spraying •OH radicals. The results show that

1.	As a strong oxidant, the •OH radicals kill different microorganisms in low concentration of 0.8 mg/L, and that is equivalent to ten thousandth the dosage of conventional chemical disinfectants (9%).
2.	With a high reaction rate constant of 109L/mol·sec, the processing time of •OH radicals is very short within 4 sec, which is one thousandth that of chemical disinfectants.
3.	The •OH spraying density is 22 μL/cm2, which is one thousandth that of other disinfectants.
4.	Observed under microscope, the •OH radicals caused irreversible damage to cells and ultimately killed them.

Acknowledgments

The work was supported by Projects of Distinguished Young and General Program from National Natural Science Foundation of China (NSFC: 61025001; 50877005), and National 863 Program from Education Ministry of China (No. 2012AA062609).