Abstract
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, hr−1) of the chamber was set to simulate fresh air ventilating dilution of indoor environment. First-order concentration decaying coefficients (Ka, min−1) 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 hr−1 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 hr−1, 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.
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
Biological contamination in agricultural and food-processing facilities
Nowadays, indoor airborne biological contamination has raised public health concerns worldwide (CitationDouwes et al., 2003; CitationSchenker et al., 1998). Exposure to high level of airborne bacterial 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 (CitationLacey and Dutkiewicz, 1994; CitationHeederik and Sigsgaard, 2005; CitationMackiewicz et al., 1999).
High airborne bacterial aerosols concentrations (up to 105 colony-forming units [CFU]/m3) for nasal breathing exposure were found in a swine confinement building investigation in Canada (CitationCormier et al., 1998; CitationCormier et al., 2000). There was 2.8 × 104 CFU/m3 of respirable airborne microorganisms recovered when Predicala et al. studied the bioaerosols concentration in the swine finishing barns (CitationPredicala 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 (CitationKim 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 (CitationLacey and Dutkiewicz, 1994; CitationRadon et al., 2002; CitationHeederik and Sigsgaard, 2005; CitationHeederik et al., 2007). In greenhouse facilities, occupational asthma and rhinitis due to the exposure to microorganisms and endotoxin during growth seasons have been reported in various investigations (CitationAdhikari et al., 2010; CitationRadon et al., 2002). High concentrations of bioaerosols and endotoxin exposure of greenhouse workers harvesting cucumbers and tomatoes were detected in Denmark (CitationMadsen 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 (CitationTsai and Liu, 2009). An investigation of a high-throughput chicken slaughter facility showed high counts of E. coli, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, andSalmonella spp. in the airborne microbial levels; controlling measures are recommended before processing materials to prevent the spread of microorganisms downstream (CitationLues et al. 2007).
Inactivating mechanism of electrolyzed water on bacteria
Facing the air quality problem caused by airborne microorganisms 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 potential (ORP) and free available chlorine (FAC) compounds (hypochlorous acid HOCl, chlorine gas Cl2, and hypochlorite ion OCl−), resulting in strong antimicrobial activity (CitationHuang et al., 2008). Studies on disinfecting mechanisms found that electrolyzed 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 (CitationZeng et al., 2010; CitationZeng et al., 2011; CitationFeliciano, Lee, and Pascall, 2012).
Neutral electrolyzed water (NEW) and acidic electrolyzed water (AEW)
The distribution of fractions of FAC compounds in electrolyzed 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 (CitationAyebah and Hung, 2005; CitationLen 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 (CitationCui et al., 2009; CitationNisola 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 (CitationAbadias 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 (CitationDeza, 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 (CitationClark et al., 2006). Escherichia coli O157:H7 food-borne pathogenic strains were spot-inoculated on lettuce leaves and were significantly reduced by NEW (CitationPangloli and Hung, 2011).
According to the investigation conducted by Monnin (CitationMonnin, 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, electrolyzed 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 (CitationGraça et al., 2011; CitationArevalos-Sánchez et al., 2012; CitationMcCarthy and Burkhardt Iii, 2012; CitationGuentzel et al., 2008; CitationZheng et al., 2012).
Despite the widely proven effectiveness bacterial contamination 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 concentrations, and nozzle orifices. With culture-based biological viability assay, the dose-response relationship between NEW and bacterial 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 The device consists 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) 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 deionized water (Milli-Q, Millipore, Billerica, MA) to FAC 50, 100, and 200 ppm as the ready-to-spray NEW disinfectant.
Figure 1. Schematic diagram of electrolyzing device.
Bacterial aerosols suspension preparation
Bacillus subtilis (BCRC 12145) vegetative cells and Escherichia 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 control studies (CitationAydogan and Gurol 2006, CitationSelkon, Babb, and Morris 1999, CitationKiura et al. 2002). Escherichia coli is a gram-negative, rod-shaped bacterium and has been applied in numerous studies on disinfection (CitationKim and Hung, 2012; CitationMonnin, Lee, and Pascall, 2012; CitationRodriguez-Garcia, Gonzalez-Romero, and Fernandez-Escartin, 2011; CitationSmigic et al., 2009; CitationPark et al., 2009). BothB. 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 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 resuspension 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 aerosol 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
schematically depicts the experimental setup for the aerosol inactivation experiment. The experimental setup comprises 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).
Figure 2. Schematic diagram of experimental setup.
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 experimental 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, hr−1). 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 hr−1. CitationElkilani and Bouhamra (2001) demonstrated that ACH rates were at 0.25–0.7 hr−1 when the HVAC system is employed, and at >1.0–1.7 hr−1 in natural ventilation conditions in general buildings. Thus, two total ACH parameters, 0.5 and 1.0 hr−1, 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 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 impactor, 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 temperature of 30 ± 1°C for 48 ± 2 hr. After incubation, the colonies formed on the plate samples were manually counted and converted to airborne bacterial concentration in CFU/m3 according to a positive-hole correction table provided by the American Conference of Industrial Hygienists (ACGIH) (CitationMacher 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 orifice 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 Equationeq 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 environment. The current Occupational Safety and Health Administration (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 concentration. 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 (CitationCode of Federal Regulations, 2012). Up to FAC 200 ppm of NEW spray was within the range of safety when tested for effectiveness to inactivate Norovirus in previous studies (CitationBolton et al., 2013; CitationPark 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 (CitationZheng et al., 2012).
The dose-response relationship between NEW disinfectant spray and bacterial aerosols was assessed with the decay coefficient of airborne survival concentration. The ventilation diluting coefficient (Kn) was defined as the first-order kinetic concentration 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:
Results
The calibration curve of bacterial aerosols in the test chamber
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 bacterial 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 about 3× 104 CFU/m3 after 80 min of delivery. Hence, the subsequent ACH natural decay and NEW inactivation experiments all applied the initial bacterial aerosols concentration at 3 × 104 CFU/m3.
Figure 3. Calibration curve of bacterial aerosols delivery in the test chamber.
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 and With the total ACH of test chamber set at 0.5 and 1.0 hr−1, the Kn values of E. coli aerosol were 0.083 and 0.135, respectively. In contrast, if the fresh air intake was shut off (total ACH = 0 hr−1), 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 hr−1, respectively. These data also revealed the same tendency found on the natural ventilation diluting effect of E. coli aerosol. Moreover, the relatively low ventilation diluting coefficient (Kn = 0.03) of both bacterial aerosols under ACH = 0 hr−1 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.
Figure 4. Ventilation dilution of E. coli aerosol in the test chamber.
Figure 5. Ventilation dilution of B. subtilis aerosol in the test chamber.
The inactivation efficiency of NEW spraying against E. coli aerosol
and 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 hr−1, 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 = 0.135, ACH = 1.0 hr−1), 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 biological decontamination capacity even under a higher ventilation 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 = 0.598, no. 8 nozzle > Ka = 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. shows inactivation efficiency of E. coli aerosol using FAC 50 and 100 ppm NEW, sprayed with the same No.8 nozzle at ACH = 1.0 hr−1. 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 applying lower FAC concentration. However, compared to Kn = 0.135 condition (ACH = 1.0 hr−1, without NEW spray intervention), the FAC 50 ppm NEW spray can still perform effective inactivation effects against E. coli aerosol.
Table 1. Ventilation dilution and inactivation efficiency of NEW spray against E. coli aerosol
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 = 1.0 hr−1).
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 = 1.0 hr−1).
The inactivation efficiency of NEW spraying against B. subtilis aerosol
and 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 hr−1. The Ka values for FAC 100 ppm NEW sprayed with no. 4 and no. 8 nozzles against B. subtilis were 0.057 and 0.063 respectively. Compared to the Kn value under the same ventilation parameter without NEW intervention (Kn = 0.045, ACH = 1.0 hr−1), 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 = 0.063, no. 8 nozzle > Ka = 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. shows the inactivation efficiency of B. subtilis aerosol using FAC 100 and 200 ppm NEW, sprayed with the same no. 8 nozzle at ACH = 1.0 hr−1. 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, compared to Kn = 0.045 condition (ACH = 1.0 hr−1, without NEW intervention), FAC 200 ppm NEW spray performs acceptable inactivation effect against B. subtilis aerosol.
Table 2. Ventilation dilution and inactivation efficiency of NEW spray against B. subtilis aerosol
Figure 8. Inactivation efficiency of B. subtilis aerosol using FAC 100 ppm NEW, sprayed with No. 4 and No. 8 nozzles in the test chamber (ACH = 1.0 hr−1).
Figure 9. Inactivation efficiency of B. subtilis aerosol using FAC 100 and 200 ppm NEW, sprayed with no. 8 nozzle in the test chamber (ACH = 1.0 hr−1).
Discussion
The capacity of NEW as an effective microbial sanitizer in food and agricultural industries has been widely tested in previous studies (CitationMonnin, Lee, and Pascall, 2012; CitationGuentzel et al., 2010; CitationPark et al., 2009; CitationCao et al., 2009; CitationArevalos-Sánchez et al., 2012; CitationDeza, Araujo, and Garrido, 2003; CitationHuang et al., 2008; CitationKoseki et al., 2001; CitationKoseki, 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 sensitive 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 setting. The B. subtilis strain is a common environmental bacteria and known to be highly resistant to various chemical and environmental 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 presented 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 = 0.135) by increasing the fresh air intake up to total ACH = 1 hr−1. However, with gram-positive B. subtilis bacterial aerosols, the Kn value was at only 0.045 when the same ACH = 1 hr−1 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 = 0.5 hr−1. CitationLee 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 = 0.5 and ACH = 1.0 hr−1. 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 bacterial 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 = 0.085 under ACH = 1 hr−1. Yet Ka = 0.598 when FAC 100 ppm NEW was sprayed against E. coli under ACH = 1 hr−1. 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 various kinds of bacterial strains in test tubes and on surfaces (CitationHuang et al., 2008; CitationKim, 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 = 1.0 hr−1 condition, the FAC 100 ppm provided an inactivating coefficient Ka = 0.598, rather than the Ka = 0.537 of FAC 50 ppm (see ). The same tendency can also be observed on B. subtilis aerosol: Ka = 0.085 could be achieved when spraying FAC 200 ppm NEW with the no. 8 nozzle under ACH = 1.0 hr−1. Lower Ka = 0.063 can be achieved in the case of parameter settings of FAC 100 ppm, no. 8 nozzle, and ACH = 1.0 hr−1 (see ). 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 inactivation 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 concentration was set at 3 × 104 CFU/m3 (which is a heavy airborne contamination, usually found in the investigation reviewed previously on bioaerosols of agricultural and food facilities). The parameters of higher ventilation rates (ACH 0.5 and 1.0 hr−1) 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 = 0.085 for FAC 200 ppm and Ka = 0.063 for FAC 100 ppm; see ).
Even considered as a nontoxic and environmentally friendly disinfectant, NEW spray still causes residual chlorine in the environment. In our study, the single spray mode was applied to understand 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 (CitationUsachev et al., 2013; CitationHsu, 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 inactivation 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 = 0.537 to E. coli aerosol Nevertheless, we doubled the FAC concentration to 100 ppm but the Ka value was at 0.452 with a no.4 nozzle spray (see ). 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 interfacial mass transfer of chlorine gas, which results in appreciable chlorine loss. CitationPark 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. CitationHsu 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 CitationHsu 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% chlorine 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 inactivation 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 bacterial 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 supporting this research under contract No. IOSH98-H311.
Related Research Data
References
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