Applying the Membrane-Less Electrolyzed Water Spraying for Inactivating Bioaerosols

The inactivating efficiency using membrane-less electrolyzed water (MLEW) spraying was evaluated against two airborne strains, Staphylococcus aureus and λ virus aerosols, in an indoor environment-simulated chamber. The air exchanged rate (ACH) of the chamber was controlled at 0.5 and 1.0 h. MLEW with a free available chlorine (FAC) concentrations of 50 and 100 ppm were pumped and sprayed into the chamber to treat microbial pre-contaminated air. Bioaerosols were collected and cultured from air before and after MLEW treatment. The first-order constant inactivation efficiency of the initial counts of 3 × 10 colony-forming units (CFU or PFU)/m for both microbial strains were observed. A higher FAC concentration of MLEW spraying resulted in greater inactivation efficiency. The inactivation coefficient under ACH 1.0 h was 0.481 and 0.554 (min) for Staphylococcus aureus of FAC 50 and 100 ppm spraying. In addition, increasing the air exchange rate also improved the inactivation rate. The inactivation coefficient of FAC 100 ppm spraying for Staphylococcus aureus was 0.412 and 0.403 (min) under ACH 1.0 and 0.5 h. These results indicated that MLEW spraying is likely to be effective in minimizing microbial airborne contamination, especially for poorly ventilated spaces.


INTRODUCTION
Individuals spend an average of 87.2% of their time indoors (Lance, 1996), highlighting the increasing importance of indoor air quality.Exposure to bioaerosols such as bacteria and its related toxin in indoor environment (Lee et al., 2012;Singh et al., 2011) may result in sick building syndrome (SBS), such as lower respiratory irritation, allergic rhinitis, couch and asthma.(Eduard et al., 1993;Koskinen et al., 1994;Melbostad et al., 1994).The airborne transmission of nosocomial bacteria not only poses health risk on patients but also impact occupational safety in health-care settings.The need to control bioaerosols in indoor environments has led to numerous strategies and technologies in the past decades.Therefore, an increasing number of air-cleaning technologies are being adopted to remove indoor bioaerosols.Currently, bioaerosol removal approaches include electret filtration (Yang and Lee, 2005;Yang et al., 2007), electrostatic precipitation (Li and Wen, 2003), ozone (Li and wang, 2003), ultraviolet germicidal irradiation (Lin and Li, 2002;Lee, 2011;Park et al., 2012), photocatalytic oxidation (Lin and Li, 2003), negative ions (Yu et al., 2006), Plasma technology (Yang et al., 2011).
Among the technologies, the generation and dissemination of chlorine-related chemical disinfectant, such as gaseous chlorine dioxide (ClO 2 ), was viewed as an effective measure to neutralize surface and airborne bacterial particles.Although chlorine dioxide is a strong and broad spectrum disinfectant, it may cause health risk on skin, eye and respiratory tract under high concentration.According to The Occupational Safety and Health Administration (OSHA) of the USA, the workplace 8 hrs time-weight average (TWA) of chlorine dioxide should below 0.3 mg/m 3 (equivalent 0.1 ppm).The 15-min short term exposure limit of chlorine dioxide should below 0.9 mg/m 3 (equivalent 0.3 ppm) (OSHA. 2008).Gaseous chlorine dioxide is more useful under dedicated delivery, en-closed ventilation, personnel-absent field such as fumigation in serious biological contaminated building.
Membrane-less Electrolyzed water (MLEW) is generated by electrolysis of saline brine in a container within anodic and cathodic electrodes without separating membrane (Clark and Barrrett, 2006;Kohno et al., 2007;Park et al., 2007;Guentzel et al., 2008;Huang et al., 2008).Its near-neutral pH product contains high oxidation-reduction potential (>1,000 mV) and free available chlorine (FAC, measuring the concentration of Cl 2 , HOCl, OCl -in liquid form) compounds.The MLEW has been reported to pose antimicrobial reactions against a variety of microorganisms in food industry and considered as alternative of traditional disinfectants.The bactericidal effect of MLEW on metal and glass surface against E.coli O157:H7, Salmonella enteritidis, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus has been performed in previous studies (Deza et al., 2005;Abadias et al., 2008).Methicillinresistant S. aureus (MRSA) and Acinetobacter baumannii were reduced with 10 6.8 fold by electrolyzed water fogging treatment while tested on ceramic tiles (Clark et al., 2007).Besides, according to the previous investigations (Guentzel et al., 2008;Arevalos-Sánchez et al., 2012;McCarthy and Burkhardt, 2012), the electrolyzed water gains the advantages of non-irritating response of mucous membranes and skin.In addition, the electrolyzed water also reveals less toxicity and environmental impact than other chemical disinfectants.Up to FAC 200 ppm of MLEW was considered as safe level (Park et al., 2007).Despite of widely used and proven effectiveness of against surface contamination, the MLEW has not been studied for the capacity to disinfectant the bioaerosols.The objective of the study is evaluating the inactivation efficiency of MLEW on bioaerosols in indoor environment, using environmental controlled chamber and cultured-based assay to determine the doseresponse relationship among species, active concentration and ventilation rate.

THEORY
The decay curves of bioaerosol concentration were more difficult to compare than the rates of bioaerosol inactivation.Therefore, bioaerosol concentration decay was analyzing using the following equations: dC/dt = -kC, (1) where C is the bioaeroosl concentration (CFU/m 3 ); C 0 and C t are the initial concentration of target bioaerosols and the concentration thereof at time t, respectively (CFU/m 3 ); t is the residence time (min); k is the decay coefficient of bioaerosol concentration (min -1 ); and, k n and k a are the decay coefficients of bioaerosol concentration associated with natural decay (physical removal) and MLEW spraying (physical removal and chemical MLEW inactivating, equal to the total eliminating), respectively (min -1 ).The coefficients of C 0 , C t and t were measured in each experiment.The decay coefficient (k) is a regression coefficient in an exponential regression analysis, specified by Eq. ( 2).The subscripts of k n and k a refer to natural decay and MLEW spraying, respectively.According to the definition of k a and k n , the decay coefficient of the k ai (only chemical MLEW inactivating) is defined as the following equation,

Generation of Membrane-less Electrolyzed Water
The MLEW used in the study was generated by handmade electrolyzing device.The schematic diagram of electrolyzing device is shown in Fig. 1.The device consists of 850 mL plastic container filled with saturated NaCl solution (6.15 M).Two 10 cm × 2 cm Pt/Ti base electrodes module was set inside the container as cathode and anode with the gap of 0.8 cm between electrodes.The current density is 25 Amp/dm 2 (ampere per decimeters, ASD) in the electrolysis container.With 30 minutes of electrolyzing process, the FAC concentration of NaCl solution would rise up to over 10,000 ppm.This 850 mL solution with high FAC concentration was then diluted with deionlized water (Milli-Q, Millipore, Billerica, MA, USA) to FAC 50 and 100 ppm as the ready-to-spray MLEW disinfectant.According to the study (Park et al. 2007), the FAC of MLEW up to 200 ppm was considered as safe level and tested to inactivate Norovirus.

Bioaerosol Preparation
Staphylococcus aureus (S. aureus, ATCC 6538) bioaerosols was chosen as the tested aerosols.Three S.
Power supply e - aureus colonies were extracted from agar plate cultures to a conical flask containing 30 mL tryptic soy broth (TSB, Bacto ™ ) with a loop.The TSB culture was then shaken at 85 rpm for 16-24 h at a temperature of 37°C.Following incubation, the TSB culture was centrifuged at 2,500 rpm for 5 mins.The supernatant was then removed, 30 mL of PBS solution (phosphate buffered saline, pH 7.2) were added and the S. aureus sediments were resuspended.Then, the osmotic pressure between the microbial cellular fluids and the buffer solution was minimized using PBS buffer solution.The above steps (except for incubation) were repeated twice to remove the TSB medium.The final PBS solution (S. aureus stock) was used for the bioaerosol generation.The concentration of viable S. aureus in the PBS solution was determined by counting colony-forming units (CFUs) on agar plates.The test bioaerosols were generated by using a Collison three-jet nebulizer (BGI Inc., Waltham, MA).The generated bioaerosols were dried by the diffusion dryer.The dried bioaerosols then passed through a Krypton 85 (Kr-85, model 3077, TSI Inc.) radioactive source, which neutralized them to the Boltzmann charge equilibrium.After it had passed through the neutralizer, the tested aerosol was delivered into testing chamber to determine initial concentration.The airborne S. aureus bacterial concentration was measured by an Andersen single-stage impactor sampler (Andersen Samplers, Inc., Atlanta, GA, USA) supplied with TSA plates (BBL Trypticase™ Soy Agar, BD, NJ, USA).The Anderson single-stage impactor has a 50% cut point size of 0.58 μm.The impactor was recommended by American Conference of Governmental Industrial Hygienists (ACGIH, 1999) and Taiwan Environmental Protection Agency (Taiwan EPA, 2008) to collect viable bacteria aerosols.The sampling flow rate is about 28.3 L/min.The each sampling time is 30 seconds.
A λ virus phage (BCRC 70193) was selected as the model strain of viruses.The λ virus phage is a harmless virus with an isometric head of approximately 0.05 μm in diameter, a thin flexible tail of approximately 0.15 μm in length, and has a diameter of approximately 7 nm.The host of the λ virus phage is E. coli K12S (BCRC 14894), and the phage was evaluated as a prospective surrogate for water-borne and food-borne viruses. 1 mL of a 16 hours E. coli K12S (as host cell) TSB culture was placed into each TSA plate and laid aside for 2 hours (each experimental set required six plates).The redundant E. coli K12S TSB culture was removed from the surface of the TSA plates, and 100 μL of viral stock were added to each plate.A sterile bent glass rod was used to spread the host culture and the λ virus phage stock uniformly over the surface of the agar plates.All plates were incubated for 8-16 h at a temperature of 37°C.Following incubation, 6 mL of sterilized distilled water were placed into each plate, and the plates were shaken at 50-60 rpm for 5 min.The supernatant was centrifuged at 10,000 rpm for 10 min and filtered with a 0.22 μm Millex GS filter unit (Millipore Corporation, Carrigtwohill, Co. Cork, Ireland) to remove the host cells.The filtered liquid (λ virus phage stock) generated the bioaerosols to determine initial concentration.Finally, the infective λ virus phage concentration in the stock was enumerated by counting the plaque-forming unit (PFU) (using the double layer agar method).An AGI-30 sampler (Model 7540, ACE GLASS Inc., NJ, USA) and a sampling pump (All Field Tech, Taiwan) were used for the λ virus bioaerosols sampling.This sampler was recommended by the American Conference of Governmental Industrial Hygienists and the International Aerobiology Symposium for sampling viable microorganisms (Jensen et al., 1992), and its collection efficiency for aerosolized viruses was comparable to those of other samplers (Tseng and Li, 2005).The collection efficiency of the sampler is a function of the particle diameter and the sampling flow rate (Hogan et al., 2005).The sampling flow rate was 12.5 L/min and the sampling time was 6-8 min.Sterilized distilled water was used for sampling the aerosolized λ virus phage (Tseng and Li, 2005;Yu et al., 2008).

Experimental Set Up
Fig. 2 schematically depicts the experimental setup for the aerosol inactivation experiment.The experimental setup comprises an aerosols nebulizer, charge neutralizer, makeup air device, MLEW spraying device (see Fig. 3), bioaerosol impactor or impinger.The model bacterial strains were aerosolized from suspension into the chamber by a threejet Collison nebulizer (BGI Inc., Waltham, MA), operated at a flow rate of 2.5 L/min.The bacterial aerosol was dried by the diffusion dryer.The dried aerosol then passed through a Kr-85 radioactive source, which neutralized them to the Boltzmann charge equilibrium.After passing through the neutralizer, the aerosol was delivered into the stainlesssteel test chamber (80 × 80 × 80 cm 3 ).Similar with indoor environment, bacterial aerosols in the test chamber may be easily diluted and removed by increasing fresh air intake, result in the decay of airborne concentration.To clarify the "natural ventilation decay" effect of fresh air intake rather than disinfectant intervention, several ventilating experimental parameters of the test chamber were examined in the study.
Before experiments started, the chamber was purged and well stabilized by clean air (without aerosols; which was produced by using HEPA filtrated air) to let the inside aerosol concentration was nearly zero.Two fans and a pump were utilized to retain a stable airflow and control the number of air exchanged rate per hour (ACH, h -1 ).In this chamber system, the ACH is equal to the total airflow per volume of the tested chamber.HEPA filtrated clean air was employed as makeup air.At a fixed total airflow rate, clean air rate was changed to survey its effect on the decay behavior of bioaerosols.Two total ACH parameters, 0.5 and 1.0 h -1 , were set in experiment.The initial relative humidity inside the chamber was set at 30% by changing the ratio of flow rate of a dry gas stream to that of a humidified gas stream generated by a water vapor saturator.The relative humidity was monitored with Q-trak (Model 8550, TSI Inc., USA).The MLEW sprayed droplets were measured by the Scanning Mobility Particle Seizer (SMPS, Model 3934 TSI Inc.) in the testing chamber.The MLEW sprayed droplet diameters (CMD) from the No. 4 and No. 8 nozzles were about 0.12 and 0.2 μm.The main active chemical principal  of the MLEW is hypochlorous acid (HOCl) molecule, which is only presented in liquid phase.HOCl converts to Cl 2 once the droplet is evaporated after sprayed:

Testing chamber
In the study, we keep the high relative humidity (> 90%) inside the test chamber after MLEW spraying.The MLEW droplet can remain liquid to clarify its microbicidal efficiency.
To determining the initial airborne bioaerosol concentration, the time-concentration calibration curve of test chamber was established by continuously deliver bacterial and virus aerosols and collect samples in 30 minute interval.The natural decay constant (k n ) was defined as the first-order decay constant (k a ) of bioaerosols concentrations without using the MLEW disinfectant spraying among various ACH parameters setting in the chamber.

The Calibration Curve of Bacterial Aerosols in Test Chamber
Fig. 4 shows the calibration curve for bacterial aerosol concentration along with continuously delivery of the test chamber.The linear relationship between bacterial concentration and delivery time can be observed.For S. aureus, the aerosol concentration can reach to about 3 × 10 4 CFU/m 3 after 50 minutes delivery.In addition, the concentration of λ virus aerosol also reached about 3× 10 4 PFU/m 3 after 60 minutes delivery.Hence, the subsequent air exchanged rates (ACH 0.5 and 1.0 h -1 ) natural decay and MLEW inactivation experiments all applied the initial bacterial aerosols concentration at 3 × 10 4 CFU/m 3 and initial λ viral aerosols concentration at 3× 10 4 PFU/m 3

The Natural Decay for Bioaerosols
The bioaerosol removal effect caused by increasing ACH in the test chamber was shown in Fig. 5 and Fig. 6.Under total ACH of test chamber were set at 0, 0.5 and 1.0 h -1 , the k n values of S. aureus bioaerosol were 0.015, 0.091 and 0.145 (min -1 ), respectively.The result indicated increasing fresh air intake results in the obviously removal effect of airborne bioaerosols.For the λ virus aerosol, the the k n values were 0.013, 0.081 and 0.138 (min -1 ) under ACH of the test chamber were set at 0, 0.5 and 1.0 h -1 , respectively.These data also performed the same trend found on natural decay effect of S. aureus aerosol.Besides, the relatively low natural decay constant (k n = 0.015 and 0.013) of both bioaerosol under ACH = 0 hr -1 indicated the gravity deposition, wall loss and desiccation of aerosols were not significant in the test chamber.In the experiment of natural decay of our study, the initial relative humidity inside the chamber was set as low as 30%.Previous studies related to environmental factors suggest that the transmission efficiency and viability of viral bioaerosols decreased with increasing relative humidity (Pica and Bouvier, 2012).In general, over 90% of the bioaerosol could remain airborne for 30 minutes after delivering into test chamber.The bioaerosols concentrations were fulfilled to conduct next step of natural ventilation decay and MLEW inactivation.

The Inactivation Efficiency of MLEW Spray Against Bioaerosol
Fig. 7 shows elimination efficiency of S. aureus aerosol using FAC 100 ppm MLEW, sprayed with No. 8 nozzle, at ACH of 0.5 and 1.0 hr -1 .The k a values for FAC 100 ppm MLEW sprayed with No. 8 nozzle at ACH of 0.5 and 1.0 hr -1 against S. aureus were 0.512 and 0.554 (min -1 ), respectively.Compare to the k n value under the same parameter without MLEW intervention (k n = 0.091 and 0.145 at ACH of 0.5 and 1.0 h -1 ), the spray of MLEW perform the effective inactivating effect against S. aureus aerosol.And according to the Eq. ( 3), the k ni (inactivation coefficient) for FAC 100 ppm MLEW sprayed with No. 8 nozzle at ACH of 0.5 and 1.0 hr -1 against S. aureus were 0.421 and 409 (min -1 ).
Fig. 8 shows elimination efficiency of λ virus aerosol using FAC 100 ppm MLEW, sprayed with No. 8 nozzle, at ACH of 0.5 and 1.0 hr -1 .The k a values for FAC 100 ppm MLEW sprayed with No. 8 nozzle at ACH of 0.5 and 1.0 hr -1 against λ virus were 0.493 and 0.541 (min -1 ), respectively.Compare to the k n value under the same parameter without MLEW intervention (k n = 0.081 and 0.138 at ACH of 0.5 and 1.0 h -1 ), the spray of MLEW perform the effective inactivating effect against λ virus aerosol.And according to the Eq. ( 3), the k ni for FAC 100 ppm MLEW sprayed with No.8 nozzle at ACH of 0.5 and 1.0 hr -1 against λ virus were 0.412 and 403 (min -1 ).
For both ACH of 0.5 and 1.0 hr -1 application, the concentration of S. aureus and λ virus aerosol decreased from 3 × 10 4 to 0 CFU/m 3 and 3 × 10 4 to 0 PFU/m 3 after 20 minutes.The result indicated MLEW spray can still perform the airborne biological contamination inactivating capacity even under higher ventilation rate.According to the experimental data, it is also finding that the k n value under the testing parameters increased with the ACH.When total ACH was increased, the air exchange rate in the testing chamber increased.High air exchange resulted in significant bioaerosols decay.
According to the Eq. ( 3), the k ai value of FAC 50 ppm and 100 ppm were 0.282 and 0.301 (min -1 ).As predicted, lower inactivating efficiency was followed with lower initial FAC concentration of MLEW.The higher initial FAC concentration of MLEW leads better inactivating efficiency against bioaerosols.These trends could also been confirmed in previous studies which use various kinds of electrolyzed water disinfectant against bacteria in test tubes and surface.In the study, the initial bacterial aerosols concentration was set at 3 × 10 4 CFU/m 3 , which is heavy airborne contamination.Also, single spray mode was applied to understand the acting time and inactivating efficiency of MLEW under heave contamination condition.Multiple spray mode of MLEW could be applied to inactivate the robust microbes such as Gram positive bacteria and fungus in the future studies.

Effect of Spraying Nozzles on the Inactivation Efficiency
Fig. 11 shows inactivation efficiency of S. aureus aerosol using FAC 100 ppm MLEW, sprayed with No. 4 and No. 8 nozzles.The ACH of test chamber was set at 1.0 h -1 .The k a values for FAC 100 ppm MLEW sprayed with No. 4 and No. 8 nozzles against S. aureus were 0.453 and 0.554 (min -1 ), respectively.According to the Eq. ( 3), the k ai value for FAC 100 ppm MLEW sprayed with No. 4 and No. 8 nozzles against S. aureus were 0.308 and 0.409 (min -1 ).Fig. 12 indicates inactivation efficiency of λ virus aerosol using FAC 100 ppm MLEW, sprayed with No. 4 and No. 8 nozzles.The ACH of test chamber was set at 1.0 h -1 .The k a values for FAC 100 ppm MLEW sprayed with No. 4 and No. 8 nozzles against λ virus were 0.439 and 0.541 (min -1 ), respectively.According to the Eq. ( 3), the k ai value for FAC 100 ppm MLEW sprayed with No. 4 and No. 8 nozzles against λ virus were 0.301 and 0.403 (min -1 ).Focused in the difference of inactivating efficiency between applying No. 4 and No. 8 nozzles, better efficiency was followed with larger spray orifice diameter (No. 8 nozzle) could be found.
The result of inactivating experiment showed that spraying MLEW with No. 8 nozzle can yield better inactivating efficiency than No. 4 nozzle under the same initial FAC concentration.We sprayed FAC 50 ppm MLEW with No. 8 nozzle for S. aureus bioaerosols could yield k ai of 0.336 (min -1 ).Nevertheless, we raised FAC concentration double to 100 ppm but k ai was 0.409 with No. 4 nozzle spraying.These results implicated orifice diameter has influent inactivation efficiency more than initial FAC concentration of MLEW while inactivating S. aureus and λ virus aerosols.Smaller orifice diameter is a mechanical disturbance that generate fine size droplet that accelerate the interfacial mass transfer of chlorine gas (Cl 2 ), result in appreciable chlorine loss.Park et al. (2007) reported approximately 70% of FAC concentration was lost and 1.3 ± 0.11 pH unit was increased during deliver hypochlorous acid solution with dynamic fogger to steel and ceramic surface.In this study, MLEW was pumped with70 kg/cm 2 through both No. 4 and No. 8 nozzle.In Hsu et al. (2004) study, the smaller sprayer orifice size produced higher reduction (spraying with orifice size 1.016 mm result in 86% chlorine reduction and 0.508 mm result in 95% chlorine reduction) in chlorine concentration than larger orifices size (1.499 mm result in 81% chlorine reduction) under the same pumping pressure (all under 103 KPa pumping) was found.And the larger droplet size would be generated from the larger orifices size.According to the experimental results, the MLEW sprayed droplet diameters (CMD) from the No. 4 and No. 8 nozzles were about 0.12 and 0.2 μm.And, the MLEW disinfectant was subsequently sprayed through 4 µm orifice diameter (No. 4) and 8 µm orifice diameter (No. 8) nozzles.)The lower inactivation efficiency while using No. 4 nozzle might result from higher reduction of FAC concentration, which is the active antimicrobial principle of MLEW.However,  enclose test chamber was used to evaluate the inactivation efficiency against bioaerosols in the study.The methodology for collecting mist type of after-spray MLEW droplet to measure the pH and FAC value is needed in future research.

CONCLUSIONS
Experimental results demonstrate that the MLEW effectively inactivate S. aureus and λ virus bioaerosols from environments controlled by testing chambers.The natural decay constant (k n ) of bioaerosols depends on the ACH.The decay constant (k a ) of bioaerosols using the MLEW increases as ACH, FAC and spraying nozzle size.The inactivation capability of the MLEW was confirmed as having a major effect on bioaerosol inactivation.