Journal of the Air & Waste Management Association

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Inactivation efficiency to Bacillus subtilis and

Escherichia coli bacterial aerosols of spraying
neutral electrolyzed water

Chi-Yu Chuang , Shinhao Yang , Ming-Yih Chang , Hsiao-Chien Huang , Chin-

Hsiang Luo , Po-Chen Hung & Wei Fang

To cite this article: Chi-Yu Chuang , Shinhao Yang , Ming-Yih Chang , Hsiao-Chien Huang ,
Chin-Hsiang Luo , Po-Chen Hung & Wei Fang (2013) Inactivation efficiency to Bacillus�subtilis and
Escherichia�coli bacterial aerosols of spraying neutral electrolyzed water, Journal of the Air & Waste
Management Association, 63:12, 1447-1456, DOI: 10.1080/10962247.2013.827604

To link to this article: https://doi.org/10.1080/10962247.2013.827604

Published online: 18 Nov 2013.

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TECHNICAL PAPER

Inactivation efficiency to Bacillus subtilis and Escherichia coli bacterial

aerosols of spraying neutral electrolyzed water
Chi-Yu Chuang,1 Shinhao Yang,2,⁄ Ming-Yih Chang,3 Hsiao-Chien Huang,2
Chin-Hsiang
1
Luo,4 Po-Chen Hung,5 and Wei Fang1
Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan, Republic of China
2
Center for General Education, Toko University, Taiwan, Republic of China
3
Department of Biomechatronics Engineering, National Ilan University, Taiwan, Republic of China
4
Department of Safety, Health and Environmental Engineering, Hungkuang University, Taiwan, Republic of China
5
Institute of Occupational Safety and Health, Council of Labor Affairs, Taiwan, Republic of China
⁄Please address correspondence to: Shinhao Yang, 51 University Rd., Sec. 2, Pu-tzu City, Chia Yi County 613, Taiwan, Republic of China;
e-mail: shinhaoyang@ntu.edu.tw

The main objective of this study is to apply neutral electrolyzed water (NEW) spraying to inactivate bioaerosols. We evaluated the
inactivation efficiency of NEW applied to inactivate two airborne bacterial Escherichia coli and Bacillus subtilis aerosols inside an
environmental-controlled chamber in the study. Generated with electrolyzing 6.15 M sodium chloride brine, the NEW with free
available chlorine (FAC) concentration 50, 100, and 200 ppm was pumped with an air pressure of 70 kg/cm2 through nozzle into the
chamber to inactive E. coli and B. subtilis aerosols precontaminated air (initial counts of 3
104 colony-forming units [CFU]/m3).
Bacterial aerosols were collected and cultured from chamber before and after NEW spray. The air exchange rate (ACH, hr1) of the
chamber was set to simulate fresh air ventilating dilution of indoor environment. First-order concentration decaying coefficients
(Ka, min1) of both bacterial aerosols were measured as an index of NEW inactivation efficiency. The result shows that higher FAC
concentration of NEW spray caused better inactivation efficiency. The Ka values under ACH 1.0 hr1 were 0.537 and 0.598 for
E. coli of FAC 50 and 100 ppm spraying, respectively. The Ka values of FAC 100 ppm and 200 ppm spraying for B. subtilis were 0.063
and 0.085 under ACH 1.0 hr1, respectively. The results indicated that NEW spray is likely to be effective in inactivation of bacterial
airborne contamination. Moreover, it is observed in the study that the increase of ventilation rate and the use of a larger orifice-size
nozzle may facilitate the inactivation efficiency.

Implications: Bacterial aerosols have been implicated in deterioration of air quality and occupational health. Effective, safe, and
economic control technology is highly demanded, especially for agricultural and food industries. In the study, NEW mist spraying
performed effectively in controlling E. coli and B. subtilis modeling bioaerosols contamination. The NEW revealed its potential as an
alternative airborne disinfectant worth being discovered for improving the environmental quality in the future.

Introduction were found in a swine confinement building investigation in

Biological contamination in agricultural and
food-processing facilities
Nowadays, indoor airborne biological contamination has
raised public health concerns worldwide (Douwes et al., 2003;
Schenker et al., 1998). Exposure to high level of airborne bacter-
ial aerosols in agricultural and food-processing facilities such as
greenhouses and swine and poultry housing may cause adverse
health effects. This has become an important issue that led to
intensive investigation in recent years (Lacey and Dutkiewicz,
1994; Heederik and Sigsgaard, 2005; Mackiewicz et al., 1999).
High airborne bacterial aerosols concentrations (up to 105
colony-forming units [CFU]/m3) for nasal breathing exposure
Canada (Cormier et al., 1998; Cormier et al., 2000). There was
2.8
104 CFU/m3 of respirable airborne microorganisms recov-
ered when Predicala et al. studied the bioaerosols concentration
in the swine finishing barns (Predicala et al., 2002). An on-site
survey in swine houses in Korea found 4 log CFU/m3 for total
airborne bacteria where air quality management is demanded
(Kim et al., 2007). High density of bacterial aerosols and related
endotoxin exposure may lead to adverse health effect of poultry
workers. The workers were reported to be high-prevalence
groups for work-related eye, respiratory, and skin symptoms in
previous studies (Lacey and Dutkiewicz, 1994; Radon et al.,
2002; Heederik and Sigsgaard, 2005; Heederik et al., 2007). In
greenhouse facilities, occupational asthma and rhinitis due to the
exposure to microorganisms and endotoxin during growth

1447
Journal of the Air & Waste Management Association, 63(12):1447–1456, 2013. Copyright © 2013 A&WMA. ISSN: 1096-2247 print
DOI: 10.1080/10962247.2013.827604
1448 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456
seasons have been reported in various investigations (Adhikari
et al., 2010; Radon et al., 2002). High concentrations of bioaer-
osols and endotoxin exposure of greenhouse workers harvesting
cucumbers and tomatoes were detected in Denmark (Madsen
et al., 2009). The exposure to high-concentration bioaerosols can
also be identified in food facilities. More than 105 CFU/m3 of
bacterial aerosols concentration in a noodle factory was
observed in central Taiwan (Tsai and Liu, 2009). An investiga-
tion of a high-throughput chicken slaughter facility showed high
counts of E. coli, Bacillus cereus, Staphylococcus aureus,
Pseudomonas aeruginosa, and Salmonella spp. in the airborne
microbial levels; controlling measures are recommended before
processing materials to prevent the spread of microorganisms
downstream (Lues et al. 2007).
Inactivating mechanism of electrolyzed water on
bacteria
Facing the air quality problem caused by airborne microor-
ganisms in the agricultural and food facilities, researchers have
shown interest in the use of chemical technologies to reduce
airborne bacteria without causing harmful effects to workers,
food materials, and animals. Electrolyzed water is generated by
electrolysis of saline brine in a cell within anodic and cathodic
electrodes with or without ion-selective permeating membrane.
The electrolyzed water contains high oxidation–reduction poten-
tial (ORP) and free available chlorine (FAC) compounds (hypo-
chlorous acid HOCl, chlorine gas Cl2, and hypochlorite ion
OCl), resulting in strong antimicrobial activity (Huang et al.,
2008). Studies on disinfecting mechanisms found that electro-
lyzed water performs dehydrogenase activities on E. coli and
S. aureus to inactivate their viability. Electrolyzed water
improves bacterial membrane permeability, resulting in rapid
leakage of DNA, potassium ions, and proteins. The multiple
disinfection mechanisms of electrolyzed water made it a broad-
spectrum disinfectant (Zeng et al., 2010; Zeng et al., 2011;
Feliciano, Lee, and Pascall, 2012).
Neutral electrolyzed water (NEW) and acidic
electrolyzed water (AEW)
The distribution of fractions of FAC compounds in electro-
lyzed water is dependent on pH and affects its bactericidal
activity. Acidic electrolyzed water (AEW) is generated in the
cathode compartment of an electrolysis cell within a membrane.
It has a strong bactericidal effect on most known pathogenic
bacteria due to its low pH (2–4), high ORP (>1,000 mV), and
higher proportion of Cl2 compared to HOCl (hypochlorous acid,
with maximal bactericidal activity). Yet neutral electrolyzed
water (NEW) is generated by electrolysis in the membraneless
electrolytic cell. It produces a solution that is close to neutral (pH
6–8) an d that does not contribute as aggressively as acidic
electrolyzed water to metal surface corrosion or skin irritation
(Ayebah and Hung, 2005; Len et al., 2002). Moreover, the
membrane-less electrolytic cell is more productive, more stable
for storage, and more convenient and economic than other
expensive and expendable membrane-within electrolysis system
(Cui et al., 2009; Nisola et al., 2011).
NEW has been reported to pose creditable antimicrobial
reactions against a variety of microorganisms in agricultural
and food industries. The performance of bactericidal efficiency
of diluted NEW against E. coli O157:H7, Erwinia carotova,
Salmonella enteritidis, and Listeria monocytogenes is at 1–2
log units (Abadias et al., 2008). NEW was also revealed to be
effective sanitizer for reducing the presence of E. coli,
L. monocytogenes, P. aeruginosa, and Staphylococcus aureus
on stainless-steel and glass surfaces with 6 log CFU/50 cm2
reduction (Deza, Araujo, and Garrido, 2005). Methicillin-
resistant S. aureus (MRSA) and Acinetobacter baumannii were
inoculated on the surface of ceramic tiles and was reduced by
106.8-fold with electrolyzed water fogging treatment (Clark et al.,
2006). Escherichia coli O157:H7 food-borne pathogenic strains
were spot-inoculated on lettuce leaves and were significantly
reduced by NEW (Pangloli and Hung, 2011).
According to the investigation conducted by Monnin
(Monnin, Lee, and Pascall, 2012), E. coli K12 and L. innocua
were significantly sanitized from cutting boards by 4 log CFU/
100 m2 with NEW. In field application, as an alternative for
chemicals traditionally used for bactericidal purposes, electro-
lyzed water is gaining popularity due to its strong bactericidal
effects when used on food and equipment surfaces and its
advantages such as safety and nonirritating response of mucous
membranes and skins. In addition, the electrolyzed water also
has the advantages of being less toxic and causing less adverse
environmental impact than other chemical disinfectants (Graça
et al., 2011; Arevalos-Sánchez et al., 2012; McCarthy and
Burkhardt Iii, 2012; Guentzel et al., 2008; Zheng et al., 2012).
Despite the widely proven effectiveness bacterial contamina-
tion on food products, food processing surfaces, and non-food-
contact surfaces, NEW has not been verified for its capacity to
neutralize bioaerosols contamination. The objective of this study
is to evaluate the inactivation efficiency of NEW on bacterial
aerosols in a simulated-indoor environment. The NEW was
delivered by compressed air-pressure spray, which is usually
used for regulating heating in indoor agricultural and food-
processing facilities. The experiments on the inactivation of
bacterial aerosols were carried out in an environmental-
controlled test chamber to understand the effects of several
disinfecting factors, such as ventilation rates, FAC concentra-
tions, and nozzle orifices. With culture-based biological viability
assay, the dose-response relationship between NEW and bacter-
ial species was determined in the study.
Material and Methods
Generation of neutral electrolyzed water (NEW)
The NEW used in the study was generated by a handmade
membraneless electrolyzing device. The schematic diagram of
the electrolyzing device is shown in Figure 1. The device con-
sists of an 850-mL cylindrical polycarbonate container (height:
15 cm; diameter: 10.5 cm) filled with saturated NaCl solution
(6.15 M). A module with two Pt/Ti base electrodes (10
2 cm2)
was set inside the container as cathode and anode with a gap of
0.8 cm between electrodes. The current density is 25 A/dm2 in
the electrolyzing device. The free available chlorine (FAC)
 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456
Figure 1. Schematic diagram of electrolyzing device.
concentration of the NEW solution was quantified following the
N,N-dimethyl-p-phenylenediamine (DPD) colorimetric method,
using a portable spectrometer (DR 2800, HACH, Loveland, CO).
The pH of the of the NEW solution was measured using a pH
meter (CyberScan pH 510, Eutech Inc., Singapore).With 30 min
of electrolyzing process, FAC concentration of the NaCl solution
would rise to more than 10,000 ppm. This 850-mL solution with
high FAC concentration was subsequently diluted with deio-
nized water (Milli-Q, Millipore, Billerica, MA) to FAC 50,
100, and 200 ppm as the ready-to-spray NEW disinfectant.
Bacterial aerosols suspension preparation
Bacillus subtilis (BCRC 12145) vegetative cells and Escheri-
chia coli (BCRC 10675) were obtained from Bioresource
Collection & Research Center (BCRC, Hsinchu, Taiwan) and
used as model bacterial aerosols in the study. The gram-positive,
rod-shaped bacterium B. subtilis is commonly used as the model
of chemical-resistant bacterial strains in previous microbial con-
trol studies (Aydogan and Gurol 2006, Selkon, Babb, and Morris
1999, Kiura et al. 2002). Escherichia coli is a gram-negative,
rod-shaped bacterium and has been applied in numerous studies
on disinfection (Kim and Hung, 2012; Monnin, Lee, and Pascall,
2012; Rodriguez-Garcia, Gonzalez-Romero, and Fernandez-
Escartin, 2011; Smigic et al., 2009; Park et al., 2009). Both
B. subtilis and E. coli bacterial strains were transferred from
frozen pure culture to 30 mL tryptic soya broth (TSB, Becton
Dickinson, Franklin Lakes, NJ) and incubated at 37 1 C for 24
hr. The TSB solutions that respectively contain B. subtilis and
1449
E. coli vegetative cells were then poured individually into sterile
tubes that were centrifuged at 2,500 rpm for 15 min. Following
the centrifugal process, the supernatants of the liquids were then
removed. The resulting pellets in the tubes were resuspended
with 10 mL presterilized phosphate buffered saline (PBS, pH
7.2) as bacterial aerosol suspension. The centrifuge and resus-
pension processes were repeated twice to totally remove TSB
medium. The osmotic pressure between the microbial cellular
fluids and the buffer was minimized by adding PBS in the
aerosol suspensions. Viable bacterial concentrations of the aero-
sol suspensions were determined by 10-fold serial dilution of 0.1
mL aliquot on tryptic soya agar plate (TSA, Becton Dickinson,
Franklin Lakes, NJ), followed by incubation at 37  1 C for 24
hr. The final bacterial concentrations of the aerosols suspensions
were adjusted to 107 CFU/ml for subsequent chamber
experiments.
Description of environment-controlled test chamber
experiment
Figure 2 schematically depicts the experimental setup for the
aerosol inactivation experiment. The experimental setup com-
prises testing chamber, aerosols nebulizer, charge neutralizer,
makeup air device, compressed air-pressure NEW spray device,
and bioaerosols sampler (single-stage viable cascade BioStage
impactor, SKC, Inc., USA). The model bacterial strains were
aerosolized from suspension into the chamber by a three-jet
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 subsequently passed through a
Kr-85 radioactive source (model 3077, TSI, Inc., USA), which
neutralized NEW particles to the Boltzmann charge equilibrium.
After passing through the neutralizer, the bacterial aerosol was
delivered into the stainless-steel test chamber (inner space size of
80
80
80 cm3).
Similar to the indoor environment, bacterial aerosols in the
test chamber can be easily diluted and removed by increasing
fresh air intake, resulting in the decay of airborne concentration.
To identify the “ventilation decay” effect of fresh air intake rather
than the disinfectant intervention, several ventilation experimen-
tal parameters of the test chamber were set and examined in the
study. A mixing fan (inside the chamber) and two air pumps
(supply and return) were utilized to maintain a stable airflow and
to control the total air exchange rate (ACH, hr1). In the test
chamber, total airflow rate is the summation of airflow rate for
return and makeup air. Total ACH equals the total airflow per
volume of the test chamber. HEPA filter-treated clean air was
employed as makeup air. The American Society of Heating,
Refrigerating, and Air-Conditioning Engineers (ASHRAE)
Standard 62–2001 indicated that the lowest indoor ACH for
housing is 0.35 hr1. Elkilani and Bouhamra (2001) demon-
strated that ACH rates were at 0.25–0.7 hr1 when the HVAC
system is employed, and at >1.0–1.7 hr1 in natural ventilation
conditions in general buildings. Thus, two total ACH para-
meters, 0.5 and 1.0 hr1, were set in the experiments to simulate
the indoor air ventilating condition. 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
1450 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456
Figure 2. Schematic diagram of experimental setup.
generated by a water vapor saturator. The relative humidity
inside the chamber was monitored with Q-trak (model 8550,
TSI, Inc., USA). The bacterial aerosols in the test chamber
were collected in accordance with Taiwan Environmental
Analysis Laboratory guideline (NIEA E301.11C, Taiwan
Environmental Protection Agency). The SKC BioStage impac-
tor, loaded with tryptic soya agar plate (Bacto TSA, Becton
Dickinson, Franklin Lakes, NJ) was utilized to collect viable
bacterial aerosols. The SKC BioSatge impactor was operated at
the flow rate of 28.3 L/min for 30 sec to collect bacterial aerosol
samples from chamber. For each sampling, three replicates were
performed. The TSA plate samples were incubated at a tempera-
ture of 30  1 C for 48  2 hr. After incubation, the colonies
formed on the plate samples were manually counted and con-
verted to airborne bacterial concentration in CFU/m3 according
to a positive-hole correction table provided by the American
Conference of Industrial Hygienists (ACGIH) (Macher 1989).
To determining the initial airborne bacterial concentration
inside the test chamber, the time–concentration calibration
curve inside the test chamber was established by continuously
delivering bacterial aerosols (B. subtilis and E. coli individually)
and collecting samples in 30-min intervals with three replicates.
Bacterial aerosols inactivation method and assay of
new spraying
For the gram-negative and sensitive E. coli model bacterial
aerosol, 100 mL of FAC 50 and 100 ppm ready-to-spray NEW
(for both, pH ranged from 7.3 to 7.5) disinfectant were pumped
into the chamber with a working pressure of 70 kg/cm2. The
NEW disinfectant was subsequently passed through 4-µm ori-
fice diameter (no. 4) and 8-µm orifice diameter (no. 8) nozzles
for aerosolization and delivery into the test chamber. The particle
diameters of NEW spray mist were measured by a scanning
mobility particle sizer (SMPS; model 3934, TSI, Inc., USA) in
the test chamber. The count means that diameters (CMD, µm) of
NEW mist spraying from the no. 4 and no. 8 nozzles were about
0.12 and 0.2 µm. The main active chemical principal of the NEW
is hypochlorous acid (HOCl) molecule, which is only presented
in the liquid phase. HOCl is converted to Cl2 once the mist is
evaporated after being sprayed, shown in eq 1:
2HCIOðaqÞ þ 2Hþ þ 2e $ Cl2ðgÞ þ 2H2 O (1)
In the study, high relative humidity (>90%) was maintained
inside the test chamber. The NEW mist remained in liquid form
(droplet) in order to examine the inactivation efficiency of the
HOCl molecule rather than gaseous Cl2. Moreover, for the gram-
positive and stress-resistant B. subtilis model bacterial aerosols,
higher FAC 100 and 200 ppm NEW disinfectant were selected
for inactivation evaluation. Lower FAC 50 and 100 ppm NEW
were prepared for inactivating gram-negative and stress-sensitive
E. coli aerosol.
Because the evaporating process converts HOCl to Cl2, the
airborne residual chlorine and potential health effect have to be
considered during the phase of spraying in the working environ-
ment. The current Occupational Safety and Health Administ-
ration (OSHA) permissible exposure limit (PEL) for chlorine is 1
ppm, specified in gas phase. In this study, NEW was released in
the form of liquid droplets, rather than evaporating to gaseous
Cl2. Therefore, we used the FAC (measuring the concentration of
Cl2, HOCl, and OCl in liquid form) to determine the concen-
tration. The U.S Food and Drug Administration (FDA)-approved
chlorine-based sanitizer contains up to FAC 200 ppm as a no-
rinse sanitizer for use on food contact surfaces (Code of Federal
Regulations, 2012). Up to FAC 200 ppm of NEW spray was
within the range of safety when tested for effectiveness to inac-
tivate Norovirus in previous studies (Bolton et al., 2013; Park
et al., 2007). In an experiment located in a layer breeding house,
Zheng and his colleague applied FAC 160 mg/L of NEW spray to
reduce the dust level (Zheng et al., 2012).
The dose-response relationship between NEW disinfectant
spray and bacterial aerosols was assessed with the decay coeffi-
cient of airborne survival concentration. The ventilation diluting
 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456 1451

coefficient (Kn) was defined as the first-order kinetic concentra-
tion decaying coefficient of bacterial aerosols without using the
NEW disinfectant spray to verify the ventilation diluting effect
among various ACH parameters setting in the chamber. On the
other hand, the NEW inactivation coefficient (Ka) was defined
as the first-order kinetic concentration decaying coefficient of
bacterial aerosols while using the NEW disinfectant spray under
various ACH parameters set in the chamber. The ventilation
diluting coefficient (Kn) and NEW mist inactivating coefficient
(Ka) were analyzed by the following equations:
dC=dt ¼ kC
Ct ¼ C0 exp ðktÞ; k ¼ kn or ka
where C is the bioaerosol concentration (CFU/m3); C0 and Ct are
the initial concentration of model bacterial aerosols and the
concentration thereof at time t, respectively (CFU/m3); t is the
residence time (min) in the test chamber; and k is the decaying
coefficient of bacterial aerosol concentration (min1). The coef-
ficients of C0, Ct, and t were measured in each experiment. The
concentration decaying coefficient (k) was a regression coeffi-
cient in an exponential regression analysis, specified by eq 3.
about 3
104 CFU/m3 after 80 min of delivery. Hence, the
subsequent ACH natural decay and NEW inactivation experi-
ments all applied the initial bacterial aerosols concentration at 3

104 CFU/m3.
The ventilation diluting effect of ACH on bacterial
aerosols
The E. coli and B. subtilis bacterial aerosols dilution effect
caused by increasing ACH in the test chamber was shown
respectively in Figure 4 and Figure 5. With the total ACH of
(2) test chamber set at 0.5 and 1.0 hr1, the Kn values of E. coli
aerosol were 0.083 and 0.135, respectively. In contrast, if the
(3) fresh air intake was shut off (total ACH ¼ 0 hr1), the Kn value
of E. coli was 0.003. The result indicated that the increase of
fresh air intake brought an obvious removal effect to airborne
bacterial aerosols inside the chamber. For the B. subtilis aerosol,
the Kn values were 0.003, 0.023, and 0.045, and total ACH of the
test chamber was set at 0, 0.5, and 1.0 hr1, respectively. These
data also revealed the same tendency found on the natural

Results
The calibration curve of bacterial aerosols in the test
chamber
Figure 3 presents the calibration curve for E. coli and
B. subtilis bacterial aerosol concentration throughout continuous
delivery in the test chamber. The linear relationship between
bacterial concentration and delivery time can be observed and
indicates stable accumulation and uniform dispersion of bacter-
ial aerosol inside the test chamber. For E. coli, the aerosol
concentration can reach up to about 3
104 CFU/m3 after 50-
min delivery. The concentration of B. subtilis aerosol reached Figure 4. Ventilation dilution of E. coli aerosol in the test chamber.

Figure 3. Calibration curve of bacterial aerosols delivery in the test chamber. Figure 5. Ventilation dilution of B. subtilis aerosol in the test chamber.
1452 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456
Table 1. Ventilation dilution and inactivation efficiency of NEW spray against E. coli aerosol
Natural ventilation decay constant (Kn) and NEW spraying inactivation constant (Ka)
Bacterial aerosols ACH (hr1) NEW intervention
E. coli 0 Without
0.5 Without
1.0 Without
1.0 FAC 50 ppm
1.0 FAC 100 ppm
1.0 FAC 100 ppm
ventilation diluting effect of E. coli aerosol. Moreover, the rela-
tively low ventilation diluting coefficient (Kn ¼ 0.03) of both
bacterial aerosols under ACH ¼ 0 hr1 indicated that the gravity
deposition and wall loss of aerosols were not significant inside
the test chamber. More than 90% of the bacterial aerosol could
remain airborne for 30 min after being delivered into the test
chamber. The relatively low gravity deposition and wall loss
characteristics of bacterial aerosols in the test chamber were
achieved to conduct the next experiments of natural ventilation
decay and NEW inactivation.
The inactivation efficiency of NEW spraying against
E. coli aerosol
Table 1 and Figure 6 present the inactivation efficiency of E. coli
aerosol using FAC 100 ppm NEW, sprayed with No. 4 and No. 8
nozzles. When the total ACH of test chamber was set at 1.0 hr1,
the Ka values for FAC 100 ppm NEW sprayed with no. 4 and no. 8
nozzles against E. coli were 0.452 and 0.598, respectively.
Compared to the Kn value of E. coli under the same ventilation
parameter without NEW intervention (Kn ¼ 0.135, ACH ¼ 1.0
hr1), the spraying of NEW effectively performed the inactivation
effect against E. coli aerosol. For both no. 4 and no. 8 nozzle NEW
spray application, the concentration of E. coli aerosol decreased
from 3
104 to 0 CFU/m3 within 20 min after spraying. The result
indicated that NEW spray is capable of performing airborne
Figure 6. Inactivation efficiency of E. coli aerosol using FAC 100 ppm NEW,
sprayed with no. 4 and no. 8 nozzles in the test chamber (ACH ¼ 1.0 hr1).
Nozzle type Ka or Kn (1/min)
— 0.003
— 0.083
— 0.135
No. 8 0.537
No. 4 0.452
No. 8 0.598
biological decontamination capacity even under a higher ventila-
tion rate. As to the effects of applying no. 4 and no. 8 nozzles, better
inactivation efficiency was found in the case of using larger spray
orifice diameter (Ka ¼ 0.598, no. 8 nozzle > Ka ¼ 0.452, no. 4
nozzle). Since mist spray of FAC 100 ppm NEW to inactivate the
E. coli aerosol was effective, a lower FAC concentration of NEW
was applied in the subsequent experiment to examine the effects of
applying lower active dose that decreases environmental chlorine
residual. Figure 7 shows inactivation efficiency of E. coli aerosol
using FAC 50 and 100 ppm NEW, sprayed with the same No.8
nozzle at ACH ¼ 1.0 hr1. The inactivating coefficient Ka value of
FAC 50 ppm and 100 ppm were 0.537 and 0.598, respectively. As
predicted, lower inactivation efficiency was observed when apply-
ing lower FAC concentration. However, compared to Kn ¼ 0.135
condition (ACH ¼ 1.0 hr1, without NEW spray intervention), the
FAC 50 ppm NEW spray can still perform effective inactivation
effects against E. coli aerosol.
The inactivation efficiency of NEW spraying against
B. subtilis aerosol
Table 2 and Figure 8 present the inactivation efficiency of
B. subtilis aerosol using FAC 100 ppm NEW, sprayed with no. 4
and no. 8 nozzles. The total ACH of test chamber was set at 1.0
hr1. The Ka values for FAC 100 ppm NEW sprayed with no.
Figure 7. Inactivation efficiency of E. coli aerosol using FAC 50 and 100 ppm
NEW, sprayed with no. 8 nozzle in the test chamber (ACH ¼ 1.0 hr1).
 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456 1453

Table 2. Ventilation dilution and inactivation efficiency of NEW spray against B. subtilis aerosol

Natural ventilation decay constant (Kn) and NEW spraying inactivation constant (Ka)
Bacterial aerosols ACH (hr1) NEW intervention Nozzle type Ka or Kn (1/min)
B. subtilis 0 Without — 0.003
0.5 Without — 0.023
1.0 Without — 0.045
1.0 FAC 100 ppm No. 8 0.063
1.0 FAC 100 ppm No. 4 0.057
1.0 FAC 200 ppm No. 8 0.085

Figure 8. Inactivation efficiency of B. subtilis aerosol using FAC 100 ppm NEW, Figure 9. Inactivation efficiency of B. subtilis aerosol using FAC 100 and 200
sprayed with No. 4 and No. 8 nozzles in the test chamber (ACH ¼ 1.0 hr1). ppm NEW, sprayed with no. 8 nozzle in the test chamber (ACH ¼ 1.0 hr1).

4 and no. 8 nozzles against B. subtilis were 0.057 and 0.063
respectively. Compared to the Kn value under the same ventila-
tion parameter without NEW intervention (Kn ¼ 0.045, ACH ¼
1.0 hr1), the spray of NEW performed milder inactivation effect
against B. subtilis aerosol. When using a no. 8 nozzle, the
concentration of B. subtilis aerosol can be reduced by about
90% after 50 min. With regard to the inactivation effects of
using no. 4 and no. 8 nozzles, better inactivation efficiency
was found with larger spray orifice diameter (Ka ¼ 0.063, no.
8 nozzle > Ka ¼ 0.057, no. 4 nozzle). This result was also found
in the experiment with E. coli. Since spray of the FAC 100 ppm
NEW to inactivate the B. subtilis aerosol was not as effective as
with E. coli, the initial FAC concentration of NEW was increased
in subsequent experiment to evaluate the appropriate active dose.
Figure 9 shows the inactivation efficiency of B. subtilis aerosol
using FAC 100 and 200 ppm NEW, sprayed with the same no. 8
nozzle at ACH ¼ 1.0 hr1. The inactivation coefficient Ka
values of FAC 100 ppm and 200 ppm were 0.063 and 0.085
respectively. As predicted, better inactivation efficiency was
found with higher initial FAC concentration. However, com-
pared to Kn ¼ 0.045 condition (ACH ¼ 1.0 hr1, without
NEW intervention), FAC 200 ppm NEW spray performs accep-
table inactivation effect against B. subtilis aerosol.
Discussion
The capacity of NEW as an effective microbial sanitizer in
food and agricultural industries has been widely tested in pre-
vious studies (Monnin, Lee, and Pascall, 2012; Guentzel et al.,
2010; Park et al., 2009; Cao et al., 2009; Arevalos-Sánchez et al.,
2012; Deza, Araujo, and Garrido, 2003; Huang et al., 2008;
Koseki et al., 2001; Koseki, Isobe, and Itoh, 2004). Because
NaCl is frequently used as an industrial raw material, the NEW
has the advantage of being economic, convenient, and safe for
in situ production in the facilities. Moreover, high air pressure
spray equipment is usually established to regulate heating and
humidity in the agricultural and food-processing facilities.
Therefore, our study aims at exploring the potential of airborne
spray of NEW disinfectant to improve air quality in indoor food-
processing and agricultural facilities. Gram-positive B. subtilis
and gram-negative E. coli were selected as model strains to
represent bioaerosols contamination.
Both gram-positive and gram-negative model strains were sen-
sitive to the NEW solution contact spray treatment. In our study,
the NEW was evaluated for its performance of reducing airborne
bacterial contamination. Overall, the NEW spray showed expected
performance to inactivate bacterial aerosols under an appropriate
1454 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456
setting. The B. subtilis strain is a common environmental bacteria
and known to be highly resistant to various chemical and environ-
mental stresses. In the study, it was subjected to represent gram-
positive, environmental-stress-resistant, airborne-infectious-
potential bacteria. On the other hand, E. coli is also usually pre-
sented in a water-related environment and in the animal digestion
tract. Some pathogenic strains of E. coli raised public health
concerns in food industries and health care facilities in recent
years. Therefore, in our study, a nonpathogenic E. coli strain was
selected as the model of gram-negative, environmental-stress-
sensitive, and airborne,contamination-potential bacterial strains.
The inactivation efficiency of ventilation dilution on
bacterial aerosols
In the first part of the bacterial aerosols ventilating dilution
experiment, gram-negative bacterial aerosols, with E. coli, even
a high level of bacterial airborne contamination (3
104 CFU/
m3, in our study) of the indoor air could be efficiently diluted and
removed (Kn ¼ 0.135) by increasing the fresh air intake up to
total ACH ¼ 1 hr1. However, with gram-positive B. subtilis
bacterial aerosols, the Kn value was at only 0.045 when the same
ACH ¼ 1 hr1 parameters applied. The same finding that E. coli
aerosol was easier to remove than B. subtilis by fresh air intake
was also consistent with the results from experiments of ACH ¼
0.5 hr1. Lee et al. (2008) reported that the presence in gram-
positive B. subtilis bacteria of strong wall structure, which
mainly consists of peptidoglycan, provides strong resistance
against airflow dilution. On the other hand, the thinner and
weaker cell wall of gram-negative E. coli bacteria resulted in a
sensitive reaction to the airflow. The difference of cell wall
structures between gram-positive and gram-negative bacteria
may explain the result of easier inactivation for E. coli than
B. subtilis aerosol under both ACH ¼ 0.5 and ACH ¼ 1.0
hr1. The result implied that increasing the ACH rate of indoor
ventilation can only gain limited benefits for improving air
quality because not all bacterial populations are be easily
removed by fresh airflow intake in an indoor environment.
The inactivation efficiency of NEW spray between
bacterial species
For the second part of the inactivation efficiencies experiment
with NEW spray between gram-positive and gram-negative bac-
terial aerosols, the B. subtilis is revealed to be more resistant than
E. coli. Higher initial FAC concentration up to FAC 200 ppm
applied to disinfect B. subtilis aerosol could only achieve Ka ¼
0.085 under ACH ¼ 1 hr1. Yet Ka ¼ 0.598 when FAC 100 ppm
NEW was sprayed against E. coli under ACH ¼ 1 hr1. Gram-
positive bacterial strains revealed better chemical resistance than
gram-negative strains. This could also be explained by the lack
of the strong and rigid cellular wall structure in gram-negative
bacteria. These findings have also been revealed in previous
studies using electrolyzed water disinfectant to neutralize var-
ious kinds of bacterial strains in test tubes and on surfaces
(Huang et al., 2008; Kim, Hung, and Brackett, 2000).
The effect of FAC concentration of NEW mist on
inactivation efficiency
In our study, higher initial FAC concentrations of NEW yielded
better inactivation efficiency against bacterial aerosols were
observed. To E. coli aerosol, sprayed under the same no. 8 nozzle
and ACH ¼ 1.0 hr1 condition, the FAC 100 ppm provided an
inactivating coefficient Ka ¼ 0.598, rather than the Ka ¼ 0.537 of
FAC 50 ppm (see Table 1). The same tendency can also be observed
on B. subtilis aerosol: Ka ¼ 0.085 could be achieved when spraying
FAC 200 ppm NEW with the no. 8 nozzle under ACH ¼ 1.0 hr1.
Lower Ka ¼ 0.063 can be achieved in the case of parameter settings
of FAC 100 ppm, no. 8 nozzle, and ACH ¼ 1.0 hr1 (see Table 2).
In the study, we used the first-order concentration decay coefficient
as the index of inactivation efficiency, which was mainly calculated
by the retention time needed to neutralize the bacterial aerosol
contamination. Higher Ka coefficient indicated more rapid inacti-
vation effect when higher initial FAC concentration (as well as
active dose) spray was applied in the study.
The purpose of this study is to establish a promising and
effective active dose of NEW spray in a setting consistent with
the “heavy contamination case scenario” in the agricultural and
food facilities. Therefore, the initial bacterial aerosols concen-
tration was set at 3
104 CFU/m3 (which is a heavy airborne
contamination, usually found in the investigation reviewed pre-
viously on bioaerosols of agricultural and food facilities). The
parameters of higher ventilation rates (ACH 0.5 and 1.0 hr1)
were also set to simulate environments of agricultural facilities in
our chamber experiment. The heavy airborne contamination
condition and high ventilation rates setting in our study affected
the test results to be seemingly less satisfying than other
chlorine-related chemical disinfecting experiments on surfaces
and in test tubes, especially in the cases of chemical stress-
resistant B. subtilis bacteria (Ka ¼ 0.085 for FAC 200 ppm and
Ka ¼ 0.063 for FAC 100 ppm; see Table 1).
Even considered as a nontoxic and environmentally friendly
disinfectant, NEW spray still causes residual chlorine in the envir-
onment. In our study, the single spray mode was applied to under-
stand the relation between contact time and inactivation efficiency
of NEW under heavy contamination and ventilated condition.
Therefore, up to NEW of FAC 200 ppm has to be applied. Lower
dosage but longer exposure time of disinfectant is expected to be
more effective to control bioaerosols with multiple or continuous
spraying strategies (Usachev et al., 2013; Hsu, Huang, and Lu,
2010). Multiple (applying NEW spray several times during a single
time period) and continuous spraying strategies of NEW could be
more effective to inactivate robust microbes such as gram-positive
bacteria and fungus in agricultural facilities. These limitations are
valuable for field-disinfection applications in future studies.
The effect of spraying nozzle orifice on inactivation
efficiency
The results of experiment on inactivation efficiency showed
that spraying NEW with a no. 8 nozzle can yield better inactiva-
tion efficiency than with a no. 4 nozzle under the same initial FAC
concentration. We sprayed FAC 50 ppm NEW with a no. 8 nozzle
that could yield Ka ¼ 0.537 to E. coli aerosol Nevertheless, we
 Chuang et al. / Journal of the Air & Waste Management Association 63 (2013) 1447–1456
doubled the FAC concentration to 100 ppm but the Ka value was at
0.452 with a no.4 nozzle spray (see Table 1). The result implicated
that nozzle orifice diameter affects inactivation efficiency more
than initial FAC concentration of NEW when inactivating E. coli.
High-pressure spray with a small-orifice nozzle is a mechanical
disturbance that generates fine particles and accelerates the inter-
facial mass transfer of chlorine gas, which results in appreciable
chlorine loss. Park et al. (2007) reported that approximately 70%
of FAC concentration was lost and there was an increase of 1.3 
0.11 pH units while delivering hypochlorous acid solution with a
dynamic fogger to steel and ceramic surfaces. Hsu et al. (2004)
demonstrated that a smaller sprayer orifice size produced higher
reduction in chlorine concentration than larger orifices size during
spray electrolyzed oxidizing water. In the study of Hsu et al.
(2004), spraying electrolyzed oxidizing water with orifice size
1.016 mm resulted in 86% chlorine reduction, 0.508 mm resulted
in 95% chlorine reduction, and 1.499 mm resulted in 81% chlor-
ine reduction, all under 103 kPa air pressure pumping. In our
study, the lower inactivation efficiency while using the smaller no.
4 nozzle might result from higher reduction of FAC concentration,
which is the active antimicrobial principle of NEW.
Conclusion
This study applied the NEW spray to inactivate bacterial
aerosols in environmental-controlled test chambers. Based on
experimental data, we consider the NEW spray technology as a
safe and economic alternative disinfectant with effective inacti-
vation efficiency against bacterial aerosols, especially for indoor
agricultural and food facilities. FAC 100 ppm NEW spray is
effective to reduce airborne concentration of gram-negative bac-
terial aerosols. Facing a gram-positive bacterial aerosol, a higher
FAC concentration of NEW should be applied. The multiple
spray strategy and various biological experimental conditions
including initial bioaerosols concentration and species need to
be studied for further discoveries of the potentials of NEW in
improving indoor environmental quality.
Acknowledgment
The authors would like to thank the Institute of Occupational
Safety and Health of Republic of China for financially support-
ing this research under contract No. IOSH98-H311.
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About the Authors
Chi-Yu Chuang is a Ph.D. candidate and Wei Fang is a professor at the
Department of Bio-Industrial Mechatronics Engineering, National Taiwan
University, Taiwan, Republic of China.
Shinhao Yang is an associate professor and Hsiao-Chien Huang is an assistant
professor at the Center of General Education, Toko University, Taiwan, Republic
of China.
Ming-Yih Chang is a senior lecturer in the Department of Biomechatronics
Engineering, National Ilan University, Taiwan, Republic of China.
Chin-Hsiang Luo is an associate professor in the Department of Safety, Health
and Environmental Engineering, Hungkuang University, Taiwan, Republic of
China.
Po-Chen Hung is a senior research scientist at the Institute of Occupational Safety
and Health, Council of Labor Affairs, Taiwan, Republic of China.