Effectiveness of slightly acidic electrolyzed water on bacteria reduction: in vitro and spray evaluation

SAEW preparation

SAEW was generated by electrolysis of a mixture of aqueous dilute solution of HCl (3%) and tap water using an Apia 60 generator (Hokuty Co. Ltd., Kanagawa, Japan). The physicochemical properties of SAEW were measured immediately after production. The pH was measured using the Piccolo pH meter (Hanna Instruments Inc., Woonsocket, RI, USA). The free chlorine concentration was determined using the HI96701 Free Chlorine Photometer. The measurement range for the photometer is 0.00 to 5.00 mg/L and the resolution is 0.01 mg/L from 0.00 to 3.50 mg/L and 0.10 mg/L above 3.50 mg/L. The SAEW was used within 1 h after preparation. SAEW solutions with 0.5 mg/L (in vitro experiments) and 10 mg/L and 20 mg/L (spray experiments) were prepared by diluting SAEW in autoclaved distilled water. The free chlorine (HOCl/OCl⁻) concentration was verified three times before every experiment.

NaOCl preparation

An analytical grade NaClO solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing 2.9 g/L of available chlorine was diluted in autoclaved distilled water to obtain 0.5 mg/L of available chlorine for the experiments. The available chlorine was determined by the HI96701 Free Chlorine Photometer.

Culture preparation

Liquid-dried cultures of E. coli (NBRC 3972), P. aeruginosa (NBRC 13275) and S. epidermidis (NBRC 100911) were obtained from the Biological Resource Center (NBRC) of the National Institute of Technology and Evaluation (NITE), Japan. Microorganisms were revived according to L-dried culture reactivation procedures provided by the manufacturer: the L-dried culture ampoule was carefully and aseptically snap opened, a few drops of 0.9% NaCl solution was added using a sterile pasteur pipette, and then gently agitated for 2 min approximately. The suspension was placed in agar plates and left undisturbed at 35 °C for 24 h (bacterial stock). The culture medium placed in petri dishes consists of a mixture of 1 g of magnesium sulfate heptahydrate (MgSO₄ · 7H₂O, Nacalai Tesque, Inc., Kyoto, Japan), 10 g of hypolypepton (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan), 2 g of yeast extract B2 (Oriental Yeast, Co., Ltd., Tokyo, Japan) and 15 g of agar powder (Wako Pure Chemical Industries Ltd., Osaka, Japan), in one L of distilled water. Bacterial culture of E. coli, P. aeruginosa and S. epidermidis were prepared to conduct experiments by transferring several colonies from the bacterial stock to five mL of 0.9% NaCl solution using a sterile inoculation loop and shaken using a vortex mixer (Vortex-Genie 2; Scientific Industries Inc., Bohemia, NY, USA).

One milliliter of each bacteria suspension (approximately 8.0 log₁₀ CFU/mL) was added to nine mL of sterilized water, SAEW 0.5 mg/L, or NaOCl 0.5 mg/L. The mixture was shaken using a vortex mixer and left undisturbed for 1 min. Following treatment with SAEW, inactivation experiments were conducted by transferring one mL of each treated sample to a sterile tube containing nine mL of neutralizing buffer solution (0.5% sodium thiosulfate solution, Na₂S₂O₃). The tube was shaken using the vortex mixer. After 5 min of neutralization, the viable count of bacteria cells in each sample was determined by plating 0.1 mL of bacterial mixture either directly or after serial dilution (1:10) in sterile 0.9% NaCl solution. The plates were incubated at 35 °C for 24 h. After this period, the bacterial colony was counted and the bactericidal activity of SAEW and NaOCl evaluated. Microbial counts were expressed as log₁₀ CFU/mL sample, with the reduction in bacterial population also calculated and presented as log₁₀ CFU/mL to compare the inactivation effects of both SAEW and NaOCl. The effect of the neutralizing solution (0.5% sodium thiosulfate solution) alone was also tested on bacterial cultures to ensure that the observed bacterial reductions were solely attributed to treatment solutions and not due to sodium thiosulfate solution. Sterilized distilled water was used as control for this experiment. Each experimental case was replicated 6 times to assure repeatability. A schematic representation of the in vitro experiment is presented in Fig. 2.

Spray experiment

Spraying SAEW is potentially an effective way to decontaminate pathogenic microorganisms in the air. Experiments were conducted in a clean booth (transparent plastic clean room) 0.9 m × 0.9 m × 0.9 m in dimension, average volume of 0.729 m³, equipped with a HEPA filter on the top to allow only clean air enter to the room as shown in Fig. 3. An air sampler (MBS-1000 manufactured by Midori Anzen Co. Ltd., Shibuya-Ku, Japan) and a 90 mm diameter plastic plate agar culture medium were placed inside the cabin and set to collect 20 L of air after 20 min. A small fan (Eluteng, 120 mm) was placed and set at 1,500 rpm to assure uniform distribution of bacteria and SAEW during experiments. An electric air pump (SABLE, 200 L/min) was also installed inside the clean booth. S. epidermidis suspension (200 mL, 10⁵ CFU/mL) was prepared in a 0.9% NaCl solution and placed in a sprayer (ultrasonic humidifier, KX 80UP-VT, 90 mL/h). SAEW solution of 10 and 20 mg/L was prepared and poured in a sprayer (ultrasonic humidifier, Ottostyle Uruoi+, 300 mL/h).

After setting the system inside the clean booth and making sure that each unit could be turned on and off from outside the booth, except the air pump that was set to collect air samples after 20 min, the door was closed. The electric air pump was turned on to pump out the air inside the booth and allow clean air to enter the clean room for about 7 min. Once the air inside was clean, bacteria suspension was sprayed for 3 min at a rate of 90 mL/h (4.5 mL of bacterial suspension release). SAEW solution was sprayed following bacterial spraying in a rate of 300 mL/h for 10, 20, 30 and 60 s and was left undisturbed for 5 min before collecting air samples with the air sampler. Experiments were conducted at 25 °C and three replicate trials were done for each case.

In vitro results

The pH of stock solutions and the available chlorine concentration of sterilized distilled water, SAEW dilutions and NaOCl dilutions used for all experiments are presented in Table 1. SAEW had a pH value ranging between 5.5 and 5.8 and a free chlorine concentration of around 29 mg/L whereas NaOCl had a pH around 8.7 and an available chlorine concentration of 2,900 mg/L. Concentrated solutions of SAEW and NaOCl were used to prepare diluted solutions for experiments.

Results of bacteria inactivation using 0.5 mg/L of SAEW and 0.5 mg/L of NaOCl are presented in Table 2 for E. coli and P. aeruginosa. Results obtained in this research showed that bacterial cultures were completely inactivated when using 0.5 mg/L of SAEW and partly reduced when applying 0.5 mg/L of NaOCl. SAEW with an available chlorine concentration of 0.5 mg/L with 1 min of treatment time showed a bacterial reduction capacity of more than 4.5 log₁₀ CFU/mL for E. coli and a bacterial reduction capacity of more than 4.4 log₁₀ CFU/mL for P. aeruginosa. On the other hand, NaOCl with an available chlorine concentration of 0.5 mg/L and also treated for 1 min showed a bacterial reduction capacity of 1.3 log₁₀ CFU/mL for E. coli and a bacterial reduction capacity of 2.3 log₁₀ CFU/mL for P. aeruginosa. The available chlorine in SAEW, even at low dosage, is the most important component in reducing bacteria count. Bacterial populations of 8.4 log₁₀ CFU/mL for E. coli, and 8.5 log₁₀ CFU/mL for P. aeruginosa were found in control experiments using sterilized distilled water. Results of in vitro experiments showed that SAEW is effective at low dosage which makes it suitable to use not only at industrial levels or factories, but also at home and public places such as restaurants where hygiene must be assured. SAEW can also be considered as an alternative solution to conventional sanitizers such as NaOCl which is usually applied in higher dosage (Issa-Zacharia et al., 2010b; Koseki et al., 2004).

The difference in bactericidal activity between SAEW and NaOCl lays on the presence of hypochlorous acid (HOCl), a very effective disinfectant, rather than the total available chlorine concentration. This is true even at concentrations of less than 0.1 mg/L (Ludovici, Phillips & Jeter, 1975). SAEW consists mainly of HOCl, whereas the ion OCl⁻ is the major chlorine specie present at the pH in commercial NaOCl dilutions (50–200 mg/L of available chlorine concentrations). It is believed that HOCl’s bactericidal activity is due to its ability to diffuse through the microbial cell, causing damage to the plasma membrane and DNA, and also inhibiting enzyme activity essential for growth (Fukuzaki, 2006). Conversely, the poor germicidal activity of the OCl⁻ specie is because penetration of this ion is prevented by the hydrophobic layer of microbe’s plasma membrane (Fukuzaki, 2006).

Nan et al. (2010), Kim et al. (2019) and Kiura et al. (2002) analyzed the bacterial cellular morphology before and after treatment with solutions containing free chlorine by transmission electron microscopy. Nan et al. (2010) and Kim et al. (2019) found that E. coli cells were remarkably damaged, destroyed or deformed after exposure to SAEW with free chlorine concentrations between 10 and 60 mg/L. Kiura et al. (2002) observed damage of the outer membrane of P. aeruginosa and inactivation of cytoplasmic enzyme after exposure to free chlorine concentration solutions, which, according to this research team, are clear indicators of the bactericidal activity of free chlorine solutions. The bactericidal effect of SAEW lies in the synergy between high oxidation-reduction potential (ORP, equal to or greater than 900 mV) and HOCl. First, a series of ORP reactions take place which damage the outer and inner membrane and inactivate the defense mechanism of bacteria. Subsequently, HOCl can penetrate the bacterial cell and oxidize it, resulting in cell inactivation, inhibition of ATP generation or death (Issa-Zacharia et al., 2011).

Spray experiments

In the spray experiments, a great reduction in the population of the bacteria S. epidermidis was observed after spraying 10 and 20 mg/L of SAEW. This indicates that SAEW can be probably used as an alternative to purifying the air in food processing factories, agricultural facilities, hospitals, care homes, fruit and vegetable markets, restaurants, etc. even at lower concentrations and for short periods of time. Results presented in Figs. 4 and 5 showed a gradual decrease in bacterial population as the SAEW spraying time was increased from 10 to 60 s. The average initial bacterial concentration for experiments using 10 mg/L was 3.72 log₁₀ CFU/m³, whereas for experiments using 20 mg/L, it was 3.76 log₁₀ CFU/m³. Reduction in terms of log₁₀ CFU/m³ and percentages are presented in Table 3. From this table, a decrease of 0.06, 0.26, 0.77 and 1.00 log₁₀ CFU/m³ or 12%, 44%, 82% and 89% was observed in bacteria population after spraying 10 mg/L of SAEW for 10, 20, 30 and 60 s respectively at a 300 mL/h rate. Also, a decrease in 0.39, 0.99, 1.80 and 3.76 log₁₀ CFU/m³ or 56%, 88%, 98% and 100% was observed in bacteria population after spraying 20 mg/L of SAEW for 10, 20, 30 and 60 s respectively at a 300 mL/h rate. This indicates that SAEW can be sprayed at lower concentrations and that continuous supply is probably not required. From the overall results, it appears that 20 mg/L of SAEW for 60 s is enough to almost completely eliminate the bacteria in the air.

A few studies reported on the application of SAEW spray for the reduction of airborne bacteria in animal housing. Weichao et al. (2016) reported on the optimization of different operating parameters (nozzle orifice diameter and spray pressure) when using SAEW spray with 30 mg/L of available chlorine concentration to improve the indoor air environment in animal housing. Hao et al. (2014) studied the distribution and subsequent reduction of airborne bacteria and fungi after 30 min of SAEW spraying in tunnel ventilated layer breeding houses. They found that both bacteria and fungi population sharply decreased after 30 min of SAEW with 250 mg/L of available chlorine concentration exposure. Zheng et al. (2013) reported on the use of SAEW spray with 150–250 mg/L of available chlorine concentration in laying-hen houses for 30 min and found that the efficiency of SAEW on the inactivation of pure cultures increased with increasing available chlorine concentration. These research teams held experiments using SAEW with available chlorine concentrations from 30 to 250 mg/L and for long periods of time. Therefore, our contribution lay on the fact that SAEW spray with much lower concentration (10 and 20 mg/L of available chlorine concentration) and reduced exposure time (intermittent or not continuous application) is effective and can be applied to public places where clean air is necessary, while guaranteeing people’s health and safety needs.